Peptides which enhance transport across tissues and methods of identifying and using the same

ABSTRACT

A method of identifying a peptide which permits or facilitates the transport of an active agent through a human or animal tissue. A predetermined amount of phage from a random phage library or a preselected phage library is administered in vivo or in situ to a site in an animal, such as into the gastro-intestinal tract. At a predetermined time, the phage which is transported across a tissue barrier is harvested at a harvesting site, such as in portal or systemic blood or brain tissue, which is separated from the site of administration by the tissue barrier to select transported phage. This transported phage is amplified in a host. This cycle of events is repeated (using the transported phage produced in the most recent cycle) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the site of administration to the harvesting site. The identity of at least one peptide coded by phage in the selected phage library is determined to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/46,411, filed Nov. 8, 1996.

TECHNICAL FIELD

[0002] This invention relates to the identification of peptide sequenceswhich permit or facilitate the transport of drugs, macromolecules, orparticles, such as biodegradable nano- and microparticles, through humanor animal tissues. In particular, this invention relates to the use ofphage display libraries in a screening assay in order to determine theidentity of peptides sequences which enhance the delivery of thebacteriophage through tissue, such as epithelial cells lining thelumenal side of the gastro-intestinal tract (GIT).

BACKGROUND ART

[0003] The epithelial cells lining the lumenal side of the GIT are amajor barrier to drug delivery following oral administration. However,there are four recognized transport pathways which can be exploited tofacilitate drug delivery and transport: the transcellular, paracellular,carrier-mediated and transcytotic transport pathways. The ability of aconventional drug, peptide, protein, macromolecule or nano- ormicroparticulate system to “interact” with one of these transportpathways may result in increased delivery of that drug or particle fromthe GIT to the underlying circulation.

[0004] In the case of the receptor-mediated, carrier-mediated ortranscytotic transport pathways, some of the “uptake” signals have beenidentified. These signals include, inter alia, folic acid, whichinteracts with the folate receptor, mannose and cetylmannoside, whichinteract with the mannose receptor, and cobalamin, which interacts withIntrinsic Factor. In addition, leucine- and tyrosine-based peptidesorting motifs or internalization sequences exist, such as YSKV, FPHL,YRGV, YQTI, TEQF, TEVM, TSAF, YTRF, which facilitate uptake or targetingof proteins from the plasma membrane to endosomes. Phage displaylibraries can be screened using specific membrane receptors or bindingsites to identify peptides that bind specifically to the receptor orbinding site. The ability of certain motifs or domains of peptides orproteins to interact with specific membrane receptors, followed bycellular uptake of the protein:receptor complex may point towards thepotential application of such motifs in facilitating the delivery ofdrugs. However, the identification of peptides or peptide motifs bytheir ability to interact with specific receptor sites or carrier sites,such as sites expressed on the apical side of the epithelial sites ofthe GIT, may not be able to determine, or may not be the most effectiveway to determine, the identity of peptides capable of enhancing thetransport of an active agent, especially a drug-loaded nano- ormicroparticle, through tissues such as epithelial lining.

[0005] Non-receptor-based assays to discover particular ligands havealso been used. For instance, a strategy for identifying peptides thatalter cellular function by scanning whole cells with phage displaylibraries is disclosed in Fong et al., Drug Development Research33:64-70 (1994). However, because whole cells, rather than intact tissueor polarized cell cultures, are used for screening phage displaylibraries, this procedure does not provide information regardingsequences whose primary function includes affecting transport acrosspolarized cell layers.

[0006] Additionally, Stevenson et al., Pharmaceutical Res. 12(9), S94(1995) discloses the use of Caco-2 monolayers to screen a synthetictripeptide combinatorial library for information relating to thepermeability of di- and tri-peptides. While useful, this technique doesnot assess the ability of the disclosed di- and tri-peptides to enhancedelivery of a drug, especially a drug-loaded nano-or microparticleformulation.

[0007] Thus, there exists a need for a method of determining peptidesequences that are particularly effective in transporting drugs,including drug-loaded nano- and microparticles, across a human or animaltissue barrier.

DISCLOSURE OF THE INVENTION

[0008] The invention provides a method of identifying a peptide whichpermits or facilitates the transport of an active agent through a humanor animal tissue. A predetermined amount of phage from a random phagelibrary or preselected phage library is plated unto or brought intocontact with a first side, preferably the apical side, of a tissuesample, either in vitro, in vivo or in situ, or polarized tissue cellculture. At a predetermined time, the phage which is transported to asecond side of the tissue opposite the first side, preferably thebasolateral side, is harvested to select transported phages. Thetransported phages are amplified in a host and this cycle of events isrepeated (using the transported phages produced in the most recentcycle) a predetermined number of times, such as from zero to six times,to obtain a selected phage library containing phage which can betransported from the first side to the second side. Lastly, the sequenceof at least one random peptide coded by phage in the selected phagelibrary is determined in order to identify a peptide which permits orfacilitates the transport of an active agent through a human or animaltissue. The transported phage can be viewed as a combination of atransporter peptide (the at least one random peptide coded by the phage)associated with an active agent payload (the phage) in which thetransporter peptide facilitates the transport of the active agentthrough the tissue. Thus, the random peptides coded by phage in theselected phage library are predictively capable of facilitatingtransport of other active agents, such as dug encapsulated nano- and/ormicroparticles, through the particular tissue.

[0009] Preferably, the tissue sample derives from the duodenum, jejunum,ileum, ascending colon, transverse colon, descending colon, pelviccolon, vascular endothelium cells which line the vascular system,vascular endothelial cells which form the blood brain barrier, alveolarcells, liver, kidney, bone marrow, retinal cells of the eye or neuronaltissue. The tissue sample can be either in vitro or in vivo. Morepreferably, the tissue sample comprises epithelial cells lining thelumenal side of the GIT, such as isolated rat colon or small intestinesegments or epithelial cells lining the lumenal side of the GIT found inan open or closed loop animal model system. Other preferred tissuesamples are heart, spleen, pancrease, thymus and brain tissue.

[0010] Preferably, the polarized tissue cell culture sample is culturedfrom GIT epithelial cells, alveolar cells, endothelial cells of theblood-brain barrier, or vascular smooth muscle cells. More preferably,the polarized tissue cell culture sample is a polarized Caco-2 cellculture or a polarized T-84 cell culture.

[0011] Preferably the random phage library or selected phage library isbrought into contact with a tissue barrier in vivo or in situ in ananimal. The phage is administered to a site in the animal and isharvested at a site which is separated from the site of administrationby a tissue barrier. Preferable harvesting sites include portal blood,systemic blood, brain tissue, liver tissue, kidney tissue, bone marrowtissue, heart tissue, spleen tissue, pancreas tissue, thymus tissue,spinal tissue, neuronal tissue, retinal eye tissue, alveolar tissue,vascular smooth muscle tissue, tissue in the vascular endothelium of theblood brain barrier, tissue in the vascular endotheliumn which lines thevascular system, pelvic colon tissue, desending colon tissue, transversecolon tissue, ascending colon tissue, ilium tissue, jejunum tissue,duodenum tissue or combinations thereof.

[0012] Preferably, the active agent is a drug or a nano- ormicroparticle. More preferably, the active agent is a drug encapsulatedor drug loaded nano- or microparticle, such as a biodegradable nano- ormicroparticle, in which the peptide is physically adsorbed or coated orcovalently bonded, such as directly linked or linked via a linkingmoiety, onto the surface of the nano- or microparticle. Alternatively,the peptide can form the nano- or microparticle itself or can bedirectly conjugated to the active agent. Such conjugations includefusion proteins in which a DNA sequence coding for the peptide is fusedin-frame to the gene or cDNA coding for a therapeutic peptide orprotein, such that the modified gene codes for a recombinant fusionprotein in which the “targeting” peptide is fused to the therapeuticpeptide or protein and where the “targeting” peptide increases theabsoption of the fusion protein from the GIT.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows the phage yield (% phage transported from the apicalto basolateral medium) in the basolateral medium of polarized Caco-2cells grown on snapwells at cycles 1, 2, 3, and 4 of panning of the ×30phage display library. For each cycle, the basolateral medium wassampled both 1 hour and 24 hours post addition of phage to the apicalmedium;

[0014] FIG. 2 shows the relative binding to fixed Caco-2 cells of 100different phage isolates from the ×30 phage display library that wereobtained from the basolateral medium at completion of cycle 4 (transportfrom apical to the basolateral medium) panning of the ×30 phage displaylibrary on Caco-2 snapwells;

[0015] FIG. 3 shows the binding of the negative control phage M13mp18and the top ten binders, clones 32, 34, 39, 40, 53, 80, 84, 97, 98 and100, [each at neat, 1:25 and 1:100 dilutions] obtained from the ×30library following cycle 4 selection on Caco-2 snapwells to fixed Caco-2cells. For reference, the ELISA absorbance reading obtained with fixedCaco-2 cells which were not treated with phage is included;

[0016] FIG. 4 shows the binding of the negative control phage M13mp 18and the top ten binders, clones 32, 34, 39, 40, 53, 80, 84, 97, 98 and100, [each at neat, 1:25 and 1:100 dilutions] obtained from the ×30library following cycle 4 selection on Caco-2 snapwells to fixed Caco-2cells, but where the background absorbance reading obtained from thefixed Caco-2 cells only, to which no phage was added, has beensubtracted; and

[0017] FIG. 5 is a graphical representation of the binding of the phageclones 39, 97 and 100 and the negative control phage M13mp18, usingeither neat phage samples or the same phage diluted 1:25 and 1:100, tofixed Caco-2 cells.

MODES FOR CARRYING OUT THE INVENTION

[0018] Surprisingly, this invention discloses a method of identifyingpeptides that are capable of facilitating the delivery or transport ofan active agent such as a drug across human or animal tissues, includingwithout limitation GIT epithelial layers, alveolar cells, endothelialcells of the blood-brain barrier, vascular smooth muscle cells, vascularendothelial cells, renal epithelial cells, M cells of the Peyers Patch,and hepatocytes. Furthermore, delivery systems, e.g., nanoparticles,microparticles, liposomes, micelles, could be coated externally with, belinked to or be comprised of these “homing” peptides to permit targeteddelivery of encapsulated drugs across particular tissues. In addition,fusion proteins can be synthesized, either in vivo or in vitro, wherebythe peptide is fused in-frame to a therapeutic peptide or protein activeagent such that the peptide enhances the delivery or transport of thetherapeutic peptide or protein across the tissue.

[0019] As used herein, the term human or animal “tissue” includes,without limitation, the duodenum, jejunum, ileum, ascending colon,transverse colon, descending colon, pelvic colon, the vascularendothelium which line the vascular system, the vascular endothelialcells which form the blood brain barrier, vascular smooth muscle,alveolar, liver, kidney, bone marrow, heart, spleen, pancreas, thymus,brain, spinal, neuronal and retinal eye tissue.

[0020] As used herein, the term “polarized tissue cell culture” refersto cells cultured so as to form polarized cell layers including, withoutlimitation, cell cultures derived from GIT epithelial cells, alveolarcells, endothelial cells of the blood-brain barrier, or vascular smoothmuscle cells or any other cell type which upon tissue culturing becomespolarized or adopts morphological characteristics or (topological)structures or appendages specific to that cell type in vivo.

[0021] As used herein, the term “active agent” includes, withoutlimitation, any drug or antigen or any drug- or antigen-loaded or drug-or antigen-encapsulated nanoparticle, microparticle, liposome, ormicellar formulation capable of eliciting a biological response in ahuman or animal. Examples of drug- or antigen-loaded or drug- orantigen-encapsulated formulations include those in which the activeagent is encapsulated or loaded into nano- or microparticles, such asbiodegradable nano- or microparticles, and which have the peptideadsorbed, coated or covalently bonded, such as directly linked or linkedvia a linking moiety, onto the surface of the nanny or microparticle.Additionally, the peptide can form the nano- or microparticle itself orthe peptide can be covalently attached to the polymer or polymers usedin the production of the biodegradable nano- or microparticles ordrug-loaded or drug-encapsulated nano- or microparticles or the peptidecan be directly conjugated to the active agent. Such conjugations toactive agents include fusion proteins in which a DNA sequence coding forthe peptide is fused in-frame to the gene or cDNA coding for atherapeutic peptide or protein such that the modified gene codes for arecombinant fusion protein.

[0022] As used herein, the term “drug” includes, without limitation, anypharmaceutically active agent. Representative drugs include, but are notlimited to, peptides or proteins, hormones, analgesics, anti-migraineagents, anti-coagulant agents, anti-emetic agents, cardiovascularagents, anti-hypertensive agents, narcotic antagonists, chelatingagents, anti-anginal agents, chemotherapy agents, sedatives,anti-neoplastics, prostaglandins and antidiuretic agents. Typical drugsinclude peptides, proteins or hormones such as insulin, calcitonin,calcitonin gene regulating protein, atrial natriuretic protein, colonystimulating factor, betaseron, erythropoietin (EPO), interferons such asα, β or γ interferon, somatropin, somatotropin, somatostatin,insulin-like growth factor (somatomedins), luteinizing hormone releasinghormone (LHRH), tissue plasminogen activator (TPA), growth hormonereleasing hormone (GHRH), oxytocin, estradiol, growth hormones,leuprolide acetate, factor VIII, interleukins such as interleukin-2, andanalogues thereof; analgesics such as fentanyl, sufentanil, butorphanol,buprenorphine, levorphanol, morphine, hydromorphone, hydrocodone,oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen,paverin, and analogues thereof; anti-migraine agents such assumatriptan, ergot alkaloids, and analogues thereof; anti-coagulantagents such as heparin, hirudin, and analogues thereof; anti-emeticagents such as scopolamine, ondansetron, domperidone, metoclopramide,and analogues thereof; cardiovascular agents, anti-hypertensive agentsand vasodilators such as diltiazem, clonidine, nifedipine, verapamil,isosorbide-5-mononitrate, organic nitrates, agents used in treatment ofheart disorders, and analogues thereof; sedatives such asbenzodiazepines, phenothiozines, and analogues thereof; narcoticantagonists such as naltrexone, naloxone, and analogues thereof;chelating agents such as deferoxamine, and analogues thereof;antidiuretic agents such as desmopressin, vasopressin, and analoguesthereof; anti-anginal agents such as nitroglycerine, and analoguesthereof; anti-neoplastics such as 5-fluorouracil, bleomycin, andanalogues thereof; prostaglandins and analogues thereof; andchemotherapy agents such as vincristine, and analogues thereof.Representative drugs also include antisense oligonucleotides, genes,gene correcting hybrid oligonucleotides, ribozymes, aptamericoligonucleotides, triple-helix forming oligonucleotides, inhibitors ofsignal transduction pathways, tyrosine kinase inhibitors and DNAmodifying agents. As used herein, the term “drug” also includes, withoutlimitation, systems for gene delivery and gene therapeutics, includingviral systems for gene delivery such as adenovirus, adeono-associatedvirus, retroviruses, herpes simplex virus, sindbus virus, liposomes,cationic lipids, dendrimers, imaging agents and enzymes.

[0023] As used herein, the term “preselected phage library” refers tolibrary consisting of a subpopulation of a phage display library. Thissubpopulation is formed by initially screening against either a targetmolecule, such as a protein, receptor, enzyme, ion channel, kinase,growth factor or growth factor receptor so as to permit the selection ofa subpopulation of phages which specifically bind to the targetmolecule. Alternatively, the subpopulation can be formed by screeningagainst a target cell or cell type or tissue type, gastrointestinaltrack, blood brain barrier or other tissue or tissue barrier so as topermit the selection of a subpopulation of phages which either bindspecifically to the target cell or target cell type or target tissue ortarget tissue barrier, or which binds to and/or is transported across(or between) the target cell, target cell type or target tissue ortarget tissue barrier either in situ or in vivo. This preselected phagelibrary or subpopulation of selected phages can also be rescreenedagainst the target molecule or cell or tissue, permitting the furtherselection of a subpopulation of phages which bind to the target moleculeor target cell, target tissue or target tissue barrier or which bind toand/or is transported across the target cell, target tissue or targettissue barrier either in situ or in vivo. Such rescreening can berepeated from zero to 30 times with each successive “pre-selected phagelibrary,” generating additional pre-selected phage libraries.

[0024] As used herein, the phrase “human or animal tissue” refers toanimal tissue explicitly including human tissue.

[0025] It has previously been shown that the NH₂-terminal amino acidsequence of the absorption proteins pIII and pVIII coded by Escherichiacoli filementous bacteriophage phage such as fd, can be modified byrecombinant DNA technology to include a library of random peptidesequences of defined length (Cwirla et al., Proc. Natl. Acad. Sci. USA487:6378-6382 (1990)). Thus, a DNA library of modified phage fdsequences, coding for variable pIII or pVIII proteins can be constructedand propagated in E. coli.

[0026] This invention discloses the use of phage display libraries suchas these in a random screening approach or a preselected phage libraryor subpopulation from a phage display library in a preselected screeningapproach in order to determine the identity of peptide sequences whichenhance the delivery of the bacteriophage from either the apical tobasolateral side or the basolateral to apical side of either culturedmodel systems or in in vitro, in situ or in vivo tissue samples.Peptides that enhance the delivery from the apical to basolateral side(e.g., gut side to blood side) can be used to enhance the delivery ofactive agents in that direction. The converse holds for peptides thatenhance the delivery from the basolateral to the apical side. Forinstance, plating on the basolateral side might determine peptidesuseful for raising a mucosal immune response to an antigen administeredIV, subcutaneously, transdermally or by the opthalmic route.

[0027] The size of the random peptide sequences coded by the librariescan be of any size. The libraries can be designed to code for linearpeptides. Alternatively, the libraries can be so designed to containcysteine residues at two or more fixed positions and thus code forcyclic peptides. As discussed further below, a preferred bacteriophagefd (e.g., from libraries L3.6, L3.15, L8.15) is a filamentous phagehaving dimensions of approximately 7 nm by 500-900 nm. On its surface,the phage expresses primarily two different proteins, the gene IIIprotein, of which there are 3-5 copies per phage particle, and the geneVIII protein, of which there are approximately 2,500 copies. In thephage display system, the genes coding for either gene III or gene VIIIhave been modified to code for and express random peptide sequences of aparticular length, such as 6-mer, 15-mer and 30-mer. In addition,multiple copies of a DNA insert coding, for example, for a random 15-mersequence can facilitate the production of random peptide sequenceslonger than 15-mer. Each library represents between 10⁸ and 10⁹ or morerandom peptide sequences. As such, the phage library can simulate ananoparticle mixture in which the nanoparticles are coated withdifferent peptides of a specified length.

[0028] During the construction of phage display libraries it is possiblethat more than one DNA insert (or partial DNA inserts which may arisedue to clevage at internal restriction sites in the DNA library or DNAinsert) can be cloned into the cloning sites in gene III or gene VIII,resulting in multiple DNA inserts in the resulting vector clone. Suchclones containing multiple DNA inserts, or derivatives thereof, have thecapacity to code for longer than expected peptides, due to the presenceof the multiple DNA inserts, provided the DNA inserts are in-frame withrespect to the gene III or gene VIII reading frame and/or provided theclones contain internal DNA sequences which are prone to or suseptibleto the process of ribosomal frameshifting during translation in vivo,which in turn can restore the reading frame of the DNA insert withrespect to the translational reading frame of gene III or gene VIII,and/or provided the mRNA coded by the DNA insert is in-frame with geneIII or gene VIII and does not contain internal translational stop ortranslational termination codons, and/or provided any internaltranslational stop or termination codon(s) can be read as a readingcodon(s) by a translational suppressor molecule in vivo, such as the TAGcodon which is decoded by the SupE suppressor in E. coli as a GLN codon.

[0029] The peptides coded by triple (or multiple) DNA inserts have thecapacity to code for longer and/or more diverse peptides. Such longerpeptides have a greater capacity to adopt secondary and tertiarystructures as opposed to shorter peptides, such as a 15-mer peptide.This capacity of peptides to adopt defined secondary and/or tertiarystructures coded by those phages containing multiple or triple DNAinserts may in-turn account for the selection of these types of phagesfrom random phage display libraries during selection or panningprocedures.

[0030] Different transport mechanisms operate in epithelial cells. Sometransport mechanisms are carrier mediated, whereby a carrier or receptorwill bind to a ligand and transport the bound ligand into or through theepithelial cell. Other transport systems operate by transcytosis,whereby a carrier or receptor site will bind a ligand, the carrier:ligand complex is internalized by endocytosis and thus delivers a ligand(or drug) into or through the cell. This invention allows for thediscovery of certain peptide sequences that bind to such active carrieror transcytotic transport systems to facilitate drug delivery. However,rather than focusing on one receptor/carrier system, the inventiondiscloses the use of a blind or random or preselected screening approachin order to identify peptide sequences that interact with undefined orunknown receptor/carrier sites in tissues, such as epithelial cells, andfacilitates the delivery of bacteriophage from the apical to basolateralside of polarized cell cultures or model tissue systems. Because thesepeptide sequences can facilitate the delivery of a bacteriophage, theyare likely to be useful in the transport of drugs and particulatesystems, especially the transport of drug loaded or encapsulated nano-and microparticulate systems when coated onto the surface of the same orfusion proteins whereby the peptide is fused to a therapeutic peptide orprotein. In addition, this invention allows for the discovery of certainpeptide sequences that recognize transcellular or paracellular transportroutes or mechnisms in cultured cells or tissues and so facilitate drugdelivery by these transport pathways.

[0031] In brief, the screening approach in the in vitro context includescontacting a predetermined amount of phage from a random phage libraryor a preselected phage library with a first side of a human or animaltissue sample or polarized tissue cell culture, harvesting phage whichis transported to the opposite side of the tissue sample or culture toselect transported phage, amplifying the transported phage in a host andidentifying at least one random peptide coded by a transported phage toidentify a peptide which permits or facilitates the transport of anactive agent through a human or animal tissue. If desired, thecontacting, harvesting and amplifying steps can be repeated apredetermined number of times using the transported phage obtained inthe previous cycle. For instance, using polarized tissue cell culturesamples such as Caco-2 cells or T-84 cells or tissue extracts such asisolated rat colon segments, phage can be plated to the apical side ofthe cultured cells or tissue segments. Subsequently, at any desiredtimepoint but usually from 1 hour to 24 hours, the basolateral medium isharvested aseptically and used to reinfect a host, such as male E. colicoding for the F¹ Factor, to produce progeny. The selected phage fromcycle one can be applied to the apical side of the cultured cells ortissue segment and again the phage in the basolateral medium iscollected, titered and amplified. Repetition of this cycle allows forenrichment of phage capable of being transported from the apical tobasolateral side and thus, the % yield of phage appearing in thebasolateral medium increases as the number of cycles increase. Afterrepeating this cycle from 0 to 30 times, preferably 3 to 20 times, theDNA sequence coding for the NH₂-terminal region of the pIII or pVIIIprotein of the purified, selected, amplified phage(s) is determined topermit deduction of the amino acid sequence of the modified phage(s)which confers the advantage of transport from the apical to basolateralside of the cultured or tissue system.

[0032] Similar to the in vitro screening approach given above, thescreening approach in the in vivo context includes contacting apredetermined amount of phage from a random phage library or apreselected phage library with a first side of a tissue barrier in vivo,harvesting phage which is transported to the opposite side of the tissuebarrier to select transported phage, amplifying the transported phage ina host and identifying at least one random peptide coded by atransported phage to identify a peptide which permits or facilitates thetransport of an active agent through a human or animal tissue. Ifdesired, the contacting, harvesting and amplifying steps can be repeateda predetermined number of times using the transported phage obtained inthe previous cycle.

[0033] For instance, the phage display library can be purified such asby either polyethylene glycol precipitations or sucrose density or CsCldensity centrifugations. The purified library can then be resuspended,such as in TBS or PBS buffer, and introduced onto one side of a tissuebarrier, such as injected into the duodenum, jejunum, ileum, colon orother in vivo animal site using, for instance, a closed loop model oropen loop model. Following introduction of the library to one side of atissue barrier, samples of bodily fluids or body tissue located acrossthe tissue barrier, such as samples of the portal circulation, systemiccirculation brain tissue, heart tissue, kidney tissue spleen tissuepancreas tissue, ileal tissue and/or duodenal tissue, are withdrawn atpredetermined time points, such as 0 to 90 minutes and/or 2 to 6 hoursor more.

[0034] For instance, an aliquot of the withdrawn bodily fluid sample(e.g., blood) is used to directly infect a host, such as E. coli, inorder to confirm the presence of phage. The remaining sample isincubated, such as overnight incubation with E. coli at 37° C. withshaking. Additionally or alternatively, harvested body tissue (e.g.,brain tissue) can be cut into small pieces, resuspended in steile PBScontaining a cocktail of protease inhibitors and homogenized. Thishomegenate can be incubated, such as overnight with E. coli at 37° withshaking. The amplified phage present in either culture can be sequencedindividually to determine the identity of peptides coded by the phageor, if further enrichment is desired, can be PEG precipitated,resuspended in PBS, and can be either further PEG-precipitated or useddirectly for administration to another animal closed or open GIT loopmodel system followed by collection of bodily fluid or body tissue andsubsequent amplification of the phage transported into such circulationsystems. In this manner, administration of the phage display librarywith, if desired, repeat administration of the amplified phage to theGIT of the animal permits the selection of phage which are transportedfrom the GIT to the portal and/or systemic circulation and/or varioustissues sites of the animal.

[0035] If desired, following administration of the phage display libraryto the tissue barrier (e.g., GIT) of the animal model, the correspondingregion of the tissue barrier can be recovered at the end of theprocedures given above. This recovered tissue can be washed repeatedlyin suitable buffers, such as PBS containing protease inhibitors andhomogenized, such as in PBS containing protease inhibitors. Thehomogenate can be used to infect a host, such as E. coli, thuspermitting amplification of phages which bind tightly to the tissuebarrier (e.g., intestinal tissue). Alternatively, the recovered tissuecan be homogenized in suitable PBS buffers, washed repeatedly and thephage present in the final tissue homogenate can be amplified in E.coli. This approach permits amplification (and subsequent identificationof the associated peptides) of phages which either bind tightly to thetissue barrier (e.g., intestinal tissue) or which are internalized bythe cells of the tissue barrier (e.g., epithelial cells of theintestinal tissue). This selection approach of phage which bind totissues or which are internalized by tissues can be repeated.

[0036] Subsequently, the corresponding peptide sequences coded by theselected phages, obtained by the procedures above and identifiedfollowing DNA sequencing of the appropriate gene III or gene VIII genesof the phage, are synthesized. The binding and transport of thesynthetic peptide itself across the model cell culture or isolatedtissue system (such as colonic) permits direct assessment of thetransport characteristics of each individual peptide. In addition,fusion of the selected peptide(s) sequences with other peptides orproteins permits direct assessment of the transport of such chimericproteins or peptides across the model systems. Such chimeric proteins orpeptides can be synthesized either in vitro or by conventionalrecombinant technology techniques whereby the cDNA coding for thetransporting peptide and the cDNA coding for the drug peptide or proteinare ligated together in-frame and are cloned into an expression vectorwhich in turn will permit expression in the desired host, be itprokaryotic cells or eukaryotic cells or transgenic animals ortransgenic plants. For instance, the cDNAs coding for the modifiedNH₂-terminal region of the pIII proteins can be subcloned into the genesor cDNAs coding for selected protein molecules (e.g., calcitonin,insulin, interferons, interleukines, cytokines, EPO, colony stimulatingfactors etc.) and these modified genes or cDNAs can be expressed in E.coli or suitable mammalian cells or transgenic animals or transgenicplants. The expressed recombinant proteins can be purified and theirtranscellular, carrier-mediated, transcytotic and/or paracellulartransport across human or animal tissue can be verified. In addition,the transporting peptides can be used to coat the surface ofnanoparticulate or microparticulate drug delivery vehicles. Suchcoatings can be performed by either direct adsorption of the peptide tothe surface of the particulate system or alternatively by covalentcoupling of the peptide to the surface of the particulate system, eitherdirectly or via a linking moiety or by covalent coupling of the peptideto the polymers used in the production of nanoparticulate ormicroparticulate drug delivery vehicles, followed by the utilization ofsuch peptide:polymer conjugates in the production of nanoparticulate ormicroparticulate drug delivery vehicles.

DESCRIPTION AND PREPARATION OF PHAGE DISPLAY LIBRARIES

[0037] Three phage display libraries, identified herein as L3.6, L3.15and L8.15, were obtained from Prof. George P. Smith at the University ofMissouri-Columbia. Each library is in the vector fUSE5, which wasderived from the parent vector “fd-tet”. In the library L3.6, random6-mer libraries are expressed by the gene III of the fd bacteriophageand are displayed on all 5 copies of the resulting protein pIIIproteins. The number of transductant clones amplified is 3.7×10¹¹ andthe size of phage DNA is 9225 bases. In the library L3.15, random 15-merlibraries are expressed by the gene III of the fd bacteriophage and aredisplayed on all 5 copies of the resulting protein pIII proteins. Thenumber of transductant clones amplified (primary amplification) is3.2×10¹¹; (secondary amplification) is 12.1×10¹² and the size of phageDNA is 9252 bases. In the library L8.15, the vector has two genes VIIIin the same genome, one of which is wild type and the other of whichdisplays the foreign residues. The random 15-mer libraries are expressedby one of the two genes VIII of the fd bacteriophage and are displayedon up to approximately 300 copies of the resulting recombinant proteinpVIII proteins. This vector is called f88-4, in which the foreign 15-meris displayed on up to approximately 300 copies of protein pVIII. Thenumber of transductant clones amplified is 2.2×10¹² and the size ofphage DNA is 9273 bases.

[0038] A 30-mer phage display library, ×30, was obtained from Dr. JamieS. Scott of Simon Fraser University. The ×30 phage display library codesfor random peptide sequences 30 residues in size. This library wasconstructed in the f88.4 vector, which carries a tetracycline resistancegene and has two pVIII genes: the wild type gene and a synthetic gene.The f88.4 library has variable inserts cloned into the synthetic pVIIIgene of the f88.4 vector.

[0039] D38 and DC43 are random phage display libraries in which gene IIIcodes for random peptides of 38 and 43 residues in size, respectively.These libraries are described in McConnell et al, Molecular Diversity1:154-176 (1995), U.S. Serial No. 310,192 filed Sep. 21, 1994, U.S.Serial No. 488,161 filed Jun. 7, 1995, and WO 96/09411, which referencesare hereby incorporated by reference.

[0040] A large scale preparation of each of the bacteriophage librarieswas made in the E. coli host strain K91Kan. A single K91Kan colony wasinnoculated into a sterile 50 ml tube containing 20 ml LB broth (Yeastextract (Gibco) −1 g; Tryptone (Gibco) −2 g; NaCl −1 g; and distilledwater −200 ml) together with kanomycin (final concentration 100 μg/ml)and grown to mid log phase with 200 rpm agitation at 37° C. (OD 0.45 at600 nm). The cells were allowed to incubate with gentle shaking (100rpm, 37° C.) for 5 min to regenerate sheared F pili. The cells werepelleted by centrifugation at 2200 rpm for 10 min at room temperature,the supernatant removed and the cells gently resuspended in 20 ml 80 mMNaCl and shaken gently (100 rpm, 37° C.) for 45 min. The cells werecentrifuged again and the cell pellet was gently resuspended in 1 mlcold NAP buffer (NaCl (5 M stock) −1.6 ml; NH₄H₂PO₄ (0.5 M stock, pH7.0) −10 ml; and distilled water −88.4 ml). The cells were stored at 4°C. and remained injectable for 3-5 days.

[0041] The primary libraries were amplified by inoculating two 11 flaskscontaining 100 ml terrific broth with 1 ml of an overnight culture ofK91Kan cells (grown in LB+100 μg/ml kanamycin). This culture wasincubated at 37° C. and 200 rpm until the OD₆₀₀ of a 1:10 dilution was0.2 and then further incubated for 5 min at 37° C. and 200 rpm to allowsheared F pili to regenerate. 10 μl of the primary library was added toeach flask with continued slow shaking for 15 min. Each culture waspoured into a prewarmed 21 flask containing 11 LB+0.22 μg/mltetracycline and shaken at 200 rpm for 35 min. 1 ml of 20 mg/mltetracycline was added and 7 μl samples were removed from each flask.The flasks were replaced in an incubator with continued shakingovernight. 200 μl of various serial dilutions (10⁻⁴,10⁻⁶, 10⁻⁸, 10⁻¹⁰dilutions) of each culture were spread on LB+40 μg/ml tetracycline and100 μg/ml kanamycin plates and incubated overnight. The colonies werecounted.

[0042] Large scale purification of phage was accomplished by dividingthe culture evenly between two 500 ml centrifuge tubes and centrifugingat 5,000 rpm for 10 min at 4° C. The supernatants were transferred tofresh tubes and recentrifuged at 8,000 rpm for 10 min at 4° C. The finalcleared supernatants were poured into fresh tubes and the net volume wasnoted. 0.15 vol PEG/NaCl (PEG 8000-100 g; NaCl −116.9 g; and distilledwater −475 ml) was added and the tubes were mixed gently by inversion(×100 times) and stored on ice for >4 h (or overnight at 4° C.).Following centrifugation at 8,000 rpm for 40 min at 4° C., thesupernatant was decanted, recentrifuged briefly and residual supernatantwas removed by pipetting. 10 ml TBS (Tris HCl (pH 7.5) −0.60 g; NaCl−0.88 g; and distilled water −100 ml) was added and the tube wasincubated at 37° C. and 200 rpm for 30 min to dissolve pellet. The tubewas centrifuged briefly and the solutions from both tubes weretransferred to a single Oak Ridge tube, centrifuged at 10-15,000 rpm for10 min at 4° C. and the supernatant was removed to a fresh tube. 0.15vol PEG/NaCl was added and the phage were allowed to precipitate on icefor 1 h. The procedures from the addition of 10 ml TBS were repeated.Into a tared 30 ml Beckman polyallomer tube, 4.83 g CsCl was added, thetube retared and the phage solution was added. TBS was added to a netweight of 10.75 g (total volume 12 ml of a 31% w/w solution of CsCl,density 1.3 g/ml). A ratio of 31:69 w/w ratio is essential. Followingcentrifugation in the ultracentrifuge at 20,000 rpm and 4° C. for 48 h,the tube was illuminated from the top with a visible light source andidentify the phage band:

[0043] Phage band—upper band, approximately 5 mm, faint, blue,non-flocculent

[0044] PEG—lower band, narrow, stringy, flocculent, opaque, white

[0045] The fluid was aspirated off to 2 mm above the phage band and thephage band was withdrawn using a sterile wide aperture transfer pipetteand placed in a 26 ml polycarbonate centrifuge tube. The tube was filledto the shoulder with TBS, mixed and centrifuged at 50,000 rpm for 4 h at4° C. in the 60 Ti rotor (repeated). The pellet was dissolved in 10 mlTB S by gentle vortexing and allowed to soften overnight in the cold andrevortexed (repeated). The pellet was then dissolved in TBS (2 ml perliter of original culture) by vortexing, allowed to soften overnight at4° C. and revortexed. The tube was centrifuged briefly to drive solutionto the bottom of the tube and transferred to 1.5 ml microtubes. Sodiumazide (0.02%) can be added and the solution can be heated to 70° C. for20 min to kill residual microorganisms. Following microfuging for 1 minto clear the solution, the supernatant was transferred to sterilemicrotubes and stored at 4° C. 200 μl of a 1:100 dilution was scannedfrom 240-320 ran to determine the concentration of physical particlesand titre 10 μl of a 10⁻⁸ dilution on 10 μl of starved K91Kan cells. 200μl of the infections was spread on LB (+40 μg/ml tetracycline and 100μg/ml kanamycin) plates, incubated at 37° C. for 24 h and counted thenumber of colonies to determine the titre of infectious units in thephage stocks.

[0046] Culturing of Caco-2, T-84 Cells

[0047] The Caco-2 (ATCC designation: CCL 248; derived from a lungmetastasis of a colon carcinoma in a 72-year old male) and T-84 cells(ATCC designation: HTB 37; isolated from a primary colonic tumor in a 72year old Caucasian male) were cultured initially in 25 cm² flasks, untilthey reached confluency. T84 cells were grown in 1:1 DMEM:Ham's F12medium containing 2 mM glutamine, 15 mM HEPES, 10% fetal calf serum(FCS), 1% MEM non essential amino acids and 50U ml⁻¹ penicillin and 50μg ml streptomycin. Caco-2 cells were grown in DMEM+glutamax-1containing 10% FCS, 1% MEM non essential amino acids, 50U ml⁻¹penicillin and 50 μg ml⁻¹ streptomycin. All cells were incubated at 37°C. in 95% O₂/5% CO₂. At confluence the cells were used to seedsnapwells.

[0048] The seeding of snapwells was essentially as follows for T-94cells (a concentration of 1×10⁶ cells/1.0 ml of medium is required foreach 12 mm snapwell; a 100% confluent flask of T84 containsapproximately 8×10⁶ cells and would be sufficient to seed 8 snapwells).The flasks were trypsinised and cells were carefully resuspended, makingsure there are no clumps or air bubbles. 2.6 ml of tissue culture mediumis placed in the bottom of the wells and 0.1 ml on the filter and placedin the incubator for 10 mins at 37° C. 1.5 ml of the cell suspension wasadded to each filter, being careful not to let any fall into the bottomof the well. The filter was placed back in the incubator and checkedafter 24 hrs. The cells were routinely monitored for adequate TER usingan EVOM chopstick epithelial voltometer (WPI). In the case of Caco-2cells, the seeding of Caco-2 cells was essentially the same as for T-84cells except that they are seeded at 5×10⁵ rather than 1×10⁶cells/snapwell.

[0049] The subsequent maintenance and feeding of the cells on thesnapwells was as follows: when feeding the wells, the medium was removedfrom the basolateral side of the snapwell first. The medium was removedfrom the monolayer with a pipette being careful not to touch the filterand then 1 ml of growth medium was place onto the apical side and 2 mlof growth medium into the basolateral side. Spillages of medium on thesides of the plate outside the well were checked for and swabbed with acotton bud moistened with alcohol if necessary. Following seeding on thesnap wells, the cells were fed on a daily basis and were cultured on thesnapwells for between 21-30 days, during which time the cellsspontaneously differentiated and become polarized.

[0050] Preparation of Intact Rat Colon Mucosae Tissue

[0051] Animals are sacrificed (by carbon monoxide), the abdominal cavitywas opened and the colon was located, removed and washed in 1×Hank'sBalanced Salt Buffer (HBSS; Gibco BRL, Cat # 14065-031). The tubularsegment was cut along the mesenteric border to give a flat square pieceof tissue. The smooth muscle layer was then removed by blunt dissectionto leave an approximate 2.5 cm² patch of epithelium.

[0052] The isolated rat colonic mucosae were mounted in Side-by-sideSweetana-Grass (SG) diffusion chambers. The mounted rat colonic mucosaein the S-G chambers were used in the analysis of phage transport fromthe apical to basolateral side of the colonic tissue.

[0053] Balancing Side-by-Side Chambers

[0054] The water bath was allowed to equilibrate to 37° C. The chamberswere filled with HBSS Buffer (see below) and the electrodes are switchedon. The input-offset control knob was adjusted to zero. The system wasallowed to equilibrate for approximately 20 minutes, making sure thereadings remain at zero throughout The electrodes were switched off andHBSS solution removed. Filters containing sheets of the rat colonicepithelium were mounted on the apparatus and 10 mls of HBSS Buffer wasadded to each side simultaneously. The tissues were oxygenated with 95%O₂/5% CO₂ and the system was allowed to equilibrate for at least 30minutes. Electrodes were switched on and the knobs set to voltage clampand current. Voltage was adjusted to give a change in current ofapproximately 2-3 μA. The timer was then set to apply a voltage every 8mins and the corresponding deflected current was used to calculate TERby applying the following Ohmic relationship: R=V/I. Recordings werecommenced for at least 10 min before any phage was added.

[0055] Enzyme Linked Immuno-Sorbent Assay (ELISA) for fd-Derived Phageon Caco-2 Cells

[0056] Caco-2 cells (100 μl) were grown to confluence in 96 well tissueculture plates (2×10⁵ cells/well grown for 2 days in growth mediumcontaining DMEM/Glutamax+1% Pen/Strep, 1% MEM & 10% FCS). After two daysgrowth, 100 μl of 10% formaldehyde [Formaldehyde (38%) sterile distilledwater (1:3 vol)] was added to the confluent Caco-2 cell monolayersfollowed by incubation for 15 min at room temperature. The contents ofthe microtitre wells was emptied by inversion/flicking and the wellswere washed three times with DPBS (Dulbecco's PBS). Each well was filledwith 200 μl of 0.1% phenylhydrazine-DPBS (0.1% phenylhydrazine in DPBS)and incubated for 1 h at 37° C. Subsequently, the contents of themicrotitre wells were emptied by inversion/flicking and the wells werewashed three times with DPBS. 200 μl of 0.5% BSA in DPBS was added toeach well followed by incubation for 1 h at room temperature. Each wellwas next washed three times in 1% BPT (1% BSA, 0.05% Tween 20 in DPBS).

[0057] Phage samples (100 μl in 1% BPT) (either neat phage at 10¹⁰pfu/ml or 1:25 or 1:100 dilutions thereof) were added to the wells,followed by incubation at room temperature for 2 h. The contents of themicrotitre wells were removed by inversion/flicking and the wells werewashed five times in 1% BPT. 100 μl of horse-radish peroxidase(HRP)-anti-M 13 conjugate (HRP/anti-M13 conjugate:horseradish peroxidaseconjugated to sheep anti-M13 IgG; 1:5000 working dilution in 1% BPT;Pharmacia 27-9402-01) was added to to each well, followed by incubationfor 1 h at room temperature. The contents of the microtitre wells wereagain removed by inversion/flicking and the wells were washed five timesin 1% BPT. 200 μl of TMB substrate solution(3,3′,5′,5-tetramethylbenzidine; Microwell Peroxidase Substrate System;Kirkegaard & Perry Laboratories CN 50-76-00; prepared by mixing equalamounts of TMB Peroxidase Substrate A and Peroxidase Solution B in aglass container immediately before use) was added to each well, followedby incubation at room temperature for 20-60 min. Thereafter, absorbancereadings were read at 650 nm on a microtitre plate reader.

[0058] Processing of Harvesting Site Tissue

[0059] Harvesting site tissue, such as brain, heart, kidney, spleen,liver, pancreas, duodenum or ileum tissue, is collected from animalsfollowing introduction in vivo of the phage display library at a siteseparated from the harvesting site by a tissue barrier. The animals aresacrificed at a predetermined time following administration of thelibrary, the tissue is removed and the tissue is either processedimmediately or frozen in liquid nitrogen (stored at −80° C.) forprocessing at a later date. The tissue samples are homogenised in PBScontaining protease inhibitors and the homogenate is used to infect E.coli, thus permitting amplification of phages that were transported tothe harvesting site tissue.

[0060] Processing of Tissue Adjacent the Phage Display LibraryAdministration Site

[0061] For use in the in vivo embodiment described herein, the phagedisplay library is purified such as by either PEG precipitation or bysucrose or CsCl density centrifugation. The phage display library isresuspended in PBS (or TBS) buffer and injected into the in vivo animalsite, such as duodenum, jejunum, ileum, colon, ascending colon,transverse colon, descending colon, pelvic colon in the closed (or open)animal (rat, rabbit or other species) loop model. Followingadministration of the phage display library to the gastrointestinaltract of the animal model, and withdrawal of portal and/or systemicblood samples at predetermined time points (such as 0 min, 15 min 30min, 45 min, 60 min up to 6 hours), or incubation of the administeredphage display library in the closed (or open) loop model for apredetermined period of time, the corresponding region of the GIT trackexposed to or incubated with the phage display library can be recoveredat the end of the experiments. Following repeated washings of therecovered intestinal tissue in suitable buffers such as PBS containingprotease inhibitors, the washed tissue is homogenised in PBS containingprotease inhibitors and the homogenate is used to infect E. coli, thuspermitting amplification of phages which can bind tightly to theintestinal tissue. Alternatively, the recovered intestinal tissue can behomogenised in suitable PBS buffers, washed repeatedly and the phagepresent in the final tissue homogenate can be amplified in E. coli. Thislatter approach also permits amplification of phages which either bindtightly to the intestinal tissue or which are internalized by theepithelial cells of the intestinal tissue

[0062] Selection of Phage With Enhanced Ability to Cross CellularBarriers

[0063] A. Treatment of Tissue Culture Cell Monolayers (Snapwell Models)With Phage Display Populations

[0064] In a laminar flow cabinet, 100 μl of phage solution was mixedwith 900 μl of growth medium without antibiotic (the completerecommended medium for each cell line but with no antibiotics added) ina microfuge tube. The experiment was carried out in duplicate andincluded a control treatment containing no phage. The TER was measuredfor each snapwell, noting the age of the cells and the passage number.Only intact monolayers of recommended age were used which had expectedTER. The basolateral medium was replaced in the snapwells with mediumwithout antibiotic and the apical medium was removed. The phagesolutions and control solutions were added to the apical side of thecells and the snapwell cultures were incubated as normal. At eachharvest time point (e.g., 1 h, 5 h, 24 h after application of phage),the medium was removed from the basolateral side and stored in a sterile2 ml screwcap tube at 4° C. At each time that the basolateral medium isremoved, the medium was replaced with fresh medium without antibiotic.When the experiments are finished, the TER was measured and themonolayers were treated with Vircon disinfectant as per normal.

[0065] The phage were titrated by preparing starved cells of E. coliK91Kan and carrying out serial dilutions of phage in the (growth mediumabove) in TBS/gelatin. 10 μl of starved cells and 10 μl ofserially-diluted phage solution were mixed in a 1.5 ml microfuge tube.The phage was allowed to infect for 10 min at room temperature. Ingeneral, the following dilutions are used: Sample Dilution t = 1 h neator 10⁻¹ t = 5 h 10⁻¹, 10⁻³ t = 24 h 10⁻¹, 10⁻³ Apical/amplified 10⁻⁶,10⁻⁷, 10⁻⁸

[0066] 1 ml of LB medium containing 0.2 μg ml⁻¹ tetracycline was addedto the phage/K91Kan cell mixtures and incubated for 30 min at 37° C. 200μl of the phage/K91Kan cell mixture was spread on LB agar platescontaining 40 μg ml⁻¹ tetracycline and 100 μg/ml kanomycin and grownovernight at 37° C. For a 10⁻² dilution (10 μl into 990 μl), 200colonies on a plate represents 1×10⁷ TU ml⁻¹.

[0067] Thus, by estimating the titre of phage which was present in thebasolateral medium and by knowing the number of phage that was appliedto the apical side, an estimate of the % yield of phage transported tothe basolateral medium from the apical side can be made.

[0068] Selected phage present in the basolateral growth medium wereamplified by adding 150 μl of PEG/NaCl per 1 ml of phage solution (poolthe harvest from all the three time-points (eg. 3×2 ml=6 ml) in an OakRidge tube. The solution is mixed very well by continuously invertingfor 2-3 min and stored at 4° C. for at least 4 h. The precipitated phageis centrifuged for 15 min at 10,000 g (8,500 rpm using Beckman JA 17rotor) in a Beckman J2-MC preparative ultracentrifuge. The supernatantwas removed and recentrifuged as before for 5 min. The pellet wasresuspended in 100 μl of TBS by leaving for 5 min at room temperatureand vortexing (repeat by leaving for 15 min and vortexing again). Thesuspended phage solution was placed in an Oak Ridge tube and 100 μl ofstarved E. coli K91Kan cells were added. The phage/cell solution wasmixed gently and left at room temperature for 30 min. 20 ml of prewarmedLB medium containing tetracycline (0.2 μg ml⁻¹) and kanomycin (100μg/ml) was added and incubated at 200 rpm at 37° C. for 30 min. 10 μl ofstock tetracycline (40 mg ml⁻¹) was added to the medium and the tube wasincubated overnight. The overnight culture was centrifuged for 15 min at3440 g (5,000 rpm using Beckman JA17 rotor) in a Beckman J2-MCpreparative ultracentrifuge. The supernatant was added to a clean(preferably sterile) Oak Ridge tube and centrifuged again for 10 min at13800 g (10,000 rpm). The supernatant was placed in a clean (preferablysterile) Oak Ridge tube containing 3 ml of PEG/NaCl and mixed bycontinuous inversion for 2-3 min. Following storage at 4° C. for atleast 4 h, the tube was centrifuged for 15 min at 13800 g (10,000 rpmusing Beckman JA17 rotor) in a Beckman J2-MC preparativeultracentrifuge. The supernatant was removed and recentrifuged as abovefor 5 min at 10,000 rpm. As much supernatant as possible was removedwith a micropipette and the pellet was resuspended in 1 ml of TBS byleaving for 5 min at room temperature and vortexing. The resuspensionwas left for 15 min and vortexed again. The phage solution wastransferred to a 1.5 ml microfuge tube and vortexed again. The solutionwas centrifuged at 13,000 rpm for 30 s in a microfuge and thesupernatant was transferred to a fresh 1.5 ml microfuge tube containing150 μl PEG/NaCl. The tube was mixed by inverting for 2-3 min and storedat 4° C. for at least 1 h. Subsequently, the tube was centrifuged at13,000 rpm for 10 min in a microfuge and the supernatant was removed andrecentrifuged for 5 min. The pellet was resuspended in 100 μl of TBS byleaving for 5 min at room temperature and vortexing. The resuspensionwas left for 15 min and vortexed again. This resuspension represents thephage selected in cycle 1. One μl should be withdrawn and used fortitration to confirm that approximately 10 ⁹ TU are present.

[0069] The phage solution is now ready for a further round of selectionin the cultured T84 and Caco-2 cells, by repeating the steps above usingthe phage transported into the basolateral medium. Thus, phage selectedfrom cycle one is now reapplied to the apical side of the Caco-2 or T-84cells growing on Snapwells. In general, in each cycle the same titre ofphage is applied to the apical side of the cells growing on snapwells.At the end of each cycle the titre of phage present in the basolateralmedium at each time point is determined and these transported phage arereamplified and recycled back through the cells. Thus, the % yield ofphage which appear in the basolateral medium increases as the number ofcycles increase. At the end of cycle five, phage have been selectedwhich are preferentially transported from the apical to basolateral sideof the cultured cells, due to the random peptide sequences displayed bythe bacteriophage gene III or gene VIII protein products.

[0070] B. Treatment of Intact Rat Colon Mucosae Tissue With PhageDisplay Populations

[0071] Once the rat colonic tissue is set up as described above,approximately 1×10¹¹ phage in HBSS buffer were applied to the gut sideof the colonic tissue, after the electrodes were switched off.Subsequently, at indicated time points, the settings were changed tovoltage and amplify, the system was grounded, the medium on both the gutside and blood side of the colonic tissue were simultaneously removed,and the medium on the blood side was saved at 4° C. The original mediumpresent on the gut side was replaced onto the gut side of the mountedcolonic tissue in the S-G chambers. Simultaneously fresh HBSS buffermedium was added to the blood side, and the tissues were oxygenated with95% O₂/5% CO₂. Electrodes were switched on again and the knobs set tovoltage clamp and current. Voltage was adjusted to give a change incurrent of approximately 2-3 μA. The timer was then set to apply avoltage every 8 mins and the corresponding deflected current was used tocalculate TER by applying the following Ohmic relationship: R=V/I.

[0072] The phage post transfer across rat colon was titrat and amplifiedas follows (phage samples titred prior to and after amplification).Serial dilutions of phage (2 μl phage+18 μl TBS/gelatin) were performedin microtitre plates and 10 μl volumes of the required dilutions weretransferred to 1.5 ml microtubes. 10 μl of starved K91Kan cells wereadded to each microtube, mixed gently and incubated at room temperaturefor 10 min. 990 μl of LB+0.2 μg ml⁻¹ tetracycline were added and themicrotubes were incubated at 37° C. for 30 min. 200 μl of the culturewere spread on LB (40 μg ml⁻¹ tetracycline+100 μg ml⁻¹ kanamycin) agarplates, incubated at 37° C. overnight and the number of colonies werecounted.

[0073] The phage was amplified by adding 150 μl of PEG/NaCl to 1 ml ofphage solution (i.e., apical or basolateral HBSS buffer from chambers)in an Oak Ridge tube, mixing by inversion (×100) and incubating at 4° C.for 4 h. The tube was centrifuged at 10,000 g for 15 min (JA17 rotor,8,500 rpm) and the supernatant was decanted and recentrifuged for 5 ml.The supernatant was removed and the pellet was resuspended in 100 pi ofTBS (leave at room temperature for 5 min, vortex, leave at roomtemperature for 15 min and revortex). A 5 μl sample was retained fortitration. 100 μl of starved K91Kan cells were added to 95 μl of phagesolution, mixed gently and incubated at room temperature for 30 min. 20ml of pre-warned LB+0.2 μg ml⁻¹ tetracycline were added and the tube wasincubated at 37° C. and 200 rpm for 30 min. 10 μl of tetracycline (40 mgml⁻¹ stock) and kanomycin (final concentration of 100 μg/ml) were addedand the tube was incubated overnight at 37° C. and 200 rpm. The tube wasthen centrifuged for 15 min at 3440 g (JA17 rotor; 5,000 rpm), thesupernatant was added to a new Oak Ridge tube and recentrifuged at13,800 g (JA17 rotor; 10,000 rpm). The supernatant was transferred to anew Oak Ridge tube containing 3 ml of PEG/NaCl, mixed by inversion(×100) and incubated at 4° C. for 4 h. The tube was then centrifuged at13,800 g, the supernatant decanted and recentrifuged at 13,800 g for 5min. The pellet was resuspended in 100 μl of TBS (leave at roomtemperature for 5 min, vortex, leave at room temperature for 15 min andrevortex). The phage solution was transferred to a microtube containing150 μl of PEG/NaCl, mixed by inversion (×100) and incubated at 4° C. for1 h. The tube was microfuged for 1 min, the supernatant removed andremicrofuged. The supernatant was removed and the pellet resuspended in100 μl of TBS (leave at room temperature for 5 min, vortex, leave atroom temperature for 15 min and revortex). 2 μl of phage for was removedfor titration while the rest was stored at 4° C.

[0074] The phage solution is now ready for a further round of selectionin the S-G mounted rat colonic tissue, by repeating the steps aboveusing the phage transported into the basolateral medium. Thus, phageselected from cycle one is now reapplied to the apical or gut side ofthe S-G mounted rat colonic tissue. In general, in each cycle the sametitre of phage is applied to the gut side of the tissue. At the end ofeach cycle the titre of phage present in the basolateral medium (bloodside) at each time point is determined and these transported phage arereamplified and recycled back through the colonic tissue. Thus, the %yield of phage which appear in the basolateral medium increases as thenumber of cycles increase. At the end of cycle five or six we haveselected for phage which are preferentially transported from the apicalor gut side of the colonic tissue to blood side or basolateral side ofthe colon tissue, due to the random peptide sequences displayed by thebacteriophage gene III or gene VIII protein products.

[0075] C. Treatment of Animal Tissue Barriers in vivo With Phage DisplayPopulations

[0076] The purified phage display library (random or preselected) isdiluted to 500 μl in PBS buffer and injected into the closed (or open)intestinal loop model (e.g., rat, rabbit or other species). At time 0and at successive time points after injection, a sample of either theportal circulation or systemic circulation is withdrawn. An aliquot ofthe withdrawn blood can be incubated with E. coli, followed by platingfor phage plaques or for transduction units or for colonies where thephage codes for resistance to antibiotics such as tetracycline. Theremainder of the withdrawn blood sample (up to 150 μl) is incubated with250 μl of E. coil and 5 ml of LB medium or other suitable growth medium.The E. coli cultures are incubated overnight by incubation at 37° C. ona shaking platform. Blood samples taken at other time points (such as 15min, 30 min, 45 min, 60 min up to 6 hours) are processed in a similarmanner, permitting amplification of phages present in the portal orsystemic circulation in E. coli. at these times. Followingamplification, the amplified phage is recovered by PEG precipitation andresuspended in PBS buffer or TBS buffer. In addition, the titer of theamplified phage, before and after PEG precipitation is determined. Theamplified, PEG precipitated phage is diluted to a known phage titer(generally between 10⁸ and 10¹⁰ phage or plaque forming units per ml)and is injected into the GIT of the animal closed (or open) loop model.Blood samples are collected from portal and for systemic circulation atvarious time points and the phage transported into the blood samples areamplified in E. coli as given above for the first cycle. Subsequently,the phage are PEG precipitated, resuspended, titered, diluted andinjected into the GIT of the animal closed (or open) loop model. Thisprocedure of phage injection followed by collection of portal and/orsystemic blood samples and amplification of phage transported into theseblood samples can be repeated, for example, up to 10 times, to permitthe selection of phages which are preferentially transported from theGIT into the portal and/or systemic circulation.

[0077] Additionally or alternatively, at the conclusion of the portalblood sampling (typically within 60 ml from administration of the phagedisplay library) or systemic blood sampling (typically within 360 minfrom administration of the phage display library) or at other convenientpredetermined times, the animal can be sacrificed and the harvestingsite tissue, such as brain tissue, removed. The tissue can be processedimmediately or frozen in liquid nitrogen (stored at −80° C.) forprocessing at a later date. Following homogenization of the tissue inPBS containing protease inhibitors, serial dilutions (such as neat,10⁻², 10⁻⁴, 10 ⁻⁶ of dilutions) of the tissue homogenate are titered inE. coli. An aliquot (100 μl) of the tissue homgentate is added to 100 μlof E. coli K91Kan starved bacteria, and incubated at 37° C. for 10 minfollowed by addition of 5 ml of LB medium or other suitable growthmedium. The E. coli cultures are incubated overnight at 37° C. andserial dilutions of amplified phage are then titered in E. coli. Asabove, the recovered phage can be administered to other animals,harvesting site tissue can be collected, and the phage transported intothe tissues can be amplified repeatedly, for example, up to 10 times, topermit the selection of phages which are preferentially transported fromthe GIT into the harvesting site tissue.

EXAMPLE 1 % Yield of in Caco-2 Cells

[0078] Libraries L3.6, L3.15, L8.15 and fUSE2 (control) were screenedusing Caco-2 cells according to the procedures given above. Thepercentage yields per cycle (1 hr, 5 hr, 24 hr and total yield) and thechange in transepithelial resistance for the cycles were measured. TheTER measurements for the Caco-2 cells remained in the range 224-449Ωcm⁻². The phage yield on the basolateral side of the cell culture isreported as a percentage of the phage applied to the apical side. Sixsuccessive screening cycles were performed and 1 hr, 5 hr and 24 hrsamples of the basolateral buffer were harvested. The percentage yieldsof phage obtained per cycle in cycles 1-6 are summarized in Table 1.Usable yields were generally obtained by the 4th cycle.

EXAMPLE 2 % Yield of φ in T-84 Cells

[0079] Libraries L3.6, L3.15, L8.15 and fUSE2 (control) were screenedusing T-84 cells according to the procedures given above. The percentageyields per cycle (1 hr, 5 hr, 24 hr and total yield) and the change intransepithelial resistance for the cycles were measured. The TERmeasurements for the T-84 cells remained in the range 224-449 Ωcm⁻². Thephage yield on the basolateral side of the cell culture is reported as apercentage of the phage applied to the apical side. Four successivescreening cycles were performed and 1 hr, 5 hr and 24 hr samples of thebasolateral buffer were harvested. The percentage yields of phageobtained per cycle in cycles 14 are summarized in Table 2. Usable yieldswere generally obtained by the 4th cycle.

EXAMPLE 3 % Yield of in Isolated Colon Segments

[0080] A phage mixture comprising libraries L3.6, L3.15 and L8.15 wasscreened using isolated rat colon according to the procedures givenabove. The phage yield on the basolateral side of the tissue sample isreported as a percentage of the phage applied to the apical side. Sixsuccessive screening cycles were performed and four 1 h samples of thebasolateral buffer were harvested. Table 3 reports the % yield of +inisolated colon segments. TABLE 1 % YIELD 0F φ IN CACO-2 CELLS Time(hours) Round 1 5 24 Total Library 13.6 1   9 × 10⁻¹   9 × 10⁻¹    9 ×10⁻¹ 0.0027 2   5 × 10⁻⁴ 0.016 0.077 0.0935 3 1.56 × 10⁻⁵ 0.0625 0.140.202 4 0.132 0.44 0.0336 0.6056 5 1.64 × 10⁻⁴ 0.069 1.377 1.45 6 3.88 ×10⁻³ 5.93 × 10⁻⁴  3.04 × 10⁻³ 0.0075 Library 3.15 1  9.5 × 10⁻¹  9.5 ×10⁻⁴  9.5 × 10⁻⁴ 0.00285 2   5 × 10⁻⁴ 20 10 30.0 3  2.5 × 10⁻⁵ 1.35 ×10⁻³ 15 15.0 4 0.207 0.048 0.82 1.075 5   2 × 10⁻⁴ 0.21 2.875 3.09 61.17 × 10⁻⁵ 19.2 6.4 25.6 Library L8.15 1 0.02 0.02 0.02 0.02 2   5 ×10⁻⁴ 0.5 0.018 0.5185 3  1.4 × 10⁻³ 0.077 1.57 1.6484 4 2.84 × 10⁻⁴ 5.39× 10⁻³ 0.14 0.1456 5 2.44 × 10⁻⁴ 0.097 1.805 1.902 6 0.0142 70.5 38 108Library fUSE2 (control) in Caco-2 cells 1 0.02 0.02 0.02 0.02 2   5 ×10⁻⁴   5 × 10⁻⁴ 0.03 0.031 3 2.08 × 10⁻⁵ 2.08 × 10⁻⁵ 1.125 × 10⁻³0.001145 4   5 × 10⁻⁴   5 × 10⁻⁴    5 × 10⁻⁴ 0.0005 (?) (?) (?) 5 2.34 ×10⁻³ 0.117 0.025 0.14 6 9.39 52.5 94 155.89

[0081] TABLE 2 % YIELD OF φ IN T-84 CELLS Time (hours) Round 1 5 24Total Library 12.6 1  3.33 × 10⁻⁶ 1.66 × 10⁻⁶ 2.4 2.4 2  7.9 × 10⁻³0.277 39.68 39.957 3  9.8 × 10⁻⁵  9.8 × 10⁻⁵ 1.04 1.04 4 0.0274 0.221.05 1.30 Library 12.15 1  4.08 × 10⁻⁴  5.8 × 10⁻³ 0.016 0.0218 2 0.3420.054 1.78 2.176 3(*)  4.3 × 10⁻⁴  4.3 × 10⁻⁴ 2.28 2.28 4 0.00 8.62 6.715.32 Library L8.15 1  2.7 × 10⁻⁶  2.7 × 10⁻⁶ 2.9 × 10⁻⁴ 0.00029 2  2.6× 10⁻⁴ 0.36 13.02 13.38 3  1.06 × 10⁻⁴ 1.06 × 10⁻⁴ 0.57 0.57 4 4.495 ×10⁻³ 52.9 40.2 93.1 Library fUSE2 (control) in T-84 cells 1    1 × 10⁻³  1 × 10⁻³   1 × 10⁻³ 0.001 2 3  2.35 × 10⁻⁴ 0.046 7.6(*) 7.6(*) 4 4.001.404 0.634 6.038 5 2.4 × 10⁻⁴/3 × 10⁻⁴

[0082] TABLE 3 % YIELD OF φ IN ISOLATED COLON SEGMENTS % yield CycleTime (h) Chamber A Chamber B 1 1 4.1 × 10⁻⁶ 4.1 × 10⁻⁶ 2 0 8.2 × 10⁻⁶ 30 4.1 × 10⁻⁶ 4 0 0 Total: 4.1 × 10⁻⁶ Total: 1.6 × 10⁻⁵ 2 1 2.6 × 10⁻⁶2.3 × 10⁻⁶ 2 0 0 3 0 0 4 0 2.3 × 10⁻⁶ Total: 2.6 × 10⁻⁶ Total: 4.6 ×10⁻⁶ 3 1 1.4 × 10⁻⁴ 2.5 × 10⁻⁴ 2 8.5 × 10⁻⁵ 4.2 × 10⁻⁴ 3 7.5 × 10⁻⁵ 6.4× 10⁻⁴ 4 7.5 × 10⁻⁵ 6.5 × 10⁻⁴ Total: 3.7 × 10⁻⁴ Total: 2.0 × 10⁻³ 4 1 00 2 0 0 3 0 1.2 × 10⁻⁵ 4 0 0 Total: 0 Total: 1.2 × 10⁻⁵ 5 1 2.3 × 10⁻⁴2.1 × 10⁻³ 2 4.725 0.049 3 1.7 × 10⁻³ 1.6 × 10⁻⁵ 4 0.0675 4.2 × 10⁻⁵Total: 4.79 Total: 0.051 6 1   7 × 10⁻³ 0.024 2 2.8 × 10⁻³ 0.03 3 7.5 ×10⁻³ 0.056 4 5.6 × 10⁻³ 0.048 Total: 0.023 Total: 0.16

EXAMPLE 4 Identification of Peptide Sequences From Transported Phage inColon Tissue Segments

[0083] Thirty-six clones from randomly selected phages from the sixthcycle of screening in rat colon segments (as given in Example 3 andTable 3) were sequenced using either the gene VIII DNA sequencing primerELN71 (SEQ ID NO: 1) or the gene III DNA sequencing primer ELN77a (SEQID NO: 17), ³⁵S-dATP and the Sequenase version 2.0 DNA sequencing kit(Amersham Life Science, UK). Progressing from cycle 1 to cycle 6, thereis a bias in the selection of phage with random peptides coded by geneVIII as opposed to gene III, perhaps because the gene III coded peptidesare present between 3-5 copies/phage particle whereas the synthetic geneVIII coded peptides are present at around 300 copies per phage particle.This higher expression level may provide a valency effect and increasethe possibility of interaction with a receptor site/pathway in thetissue sample.

[0084] A number of clones/DNA sequences are present more than once,suggesting some type of preferential selection. Thus, SEQ ID NO: 2 (aClass of 9 clones −25% presence), SEQ ID NO: 3 (a Class of 5 clones−13.9% presence), SEQ ID NO: 4 (a Class of 3 −8.3% presence) weredetermined from this 36 clone sample from cycle 6. All of these Classesconsist of clones with triple DNA inserts. Individual isolates are givenby SEQ. ID. NO: 5 to SEQ ID NO: 9 (triple DNA inserts) and SEQ ID NO: 10(single insert).

[0085] Based on the recurrent random peptide sequences in these classes,two synthetic oligonucleotides were constructed and used to screen phagepopulations representing colon screening cycles 1-6 in a series ofoligonucleotide hybridization reactions to determine whether these phageand corresponding peptides were being selected during the screeningprocess. Thus, oligonucleotides ELN93 and ELN94 correspond to a partialcoding region in those phage clones for SEQ ID NO: 2 and SEQ ID NO: 3,respectively. The incidence of reactivity per screening cycle issummarized in Table 4 below. From the data presented in Table 4, itappears that there is a gradual selection of phage which hybridize tooligonucleotide ELN93 and ELN94 progressing from cycle I through cycle6. Probe reactivity is expressed as a percentage of the total number ofcolonies screened per phage population. As a control, the unselected,starting libraries (L3.6, L3. 15 and L8.15) were also included. TABLE 4HYBRIDIZATION OF PHAGE POPULATIONS (COLON SCREENING CYCLES 1-6 ANDUNSELECTED LIBRARIES L3.6, L3.15 AND L8.15) WITH OLIGONUCLEOTIDES ELN93AND ELN94 ELN93 ELN94 1 0.4 0.4 2 4.7 0 3 17.4 0 4 26.4 1.25 >20.0 >40.0 6 62.5 >40.0 L3.6 0.8 0 L3.15 0.8 0 L8.15 0.3 0

[0086] The phage populations representing Caco-2 screening cycles 1-6and T-84 screening cycles 1-4, as given above in Example 1, Table 1 andExample 2, Table 2, respectively, were also assessed for reactivity tothe oligonucleotide probes ELN93 and ELN94. The incidence of reactivityper screening cycle in Caco-2 and T-84 cells is compared to reactivityin colon tissue in Table 5 (ELN93) and Table 6 (ELN94). In these Tables,probe reactivity is expressed as a percentage of the total number ofcolonies screened per phage population. Some reactivity was detected inCaco-2 selected clones using ELN93. The gradual selection of ELN93reactive phage during progression from cycles 1 to 6 observed for phagelibrary L3.15B correlated with the pattern of reactivity previouslyobserved for colon-selected phage although the overall reactivityachieved was substantially lower. ELN94 reactivity was identified inboth Caco-2 and T-84 selected clones. Increasing reactivity from cycles1 to 6 was observed for Caco-2 selected libraries L3.6B, L3.15B andL8.15B as well as the T-84 selected library L3.15A. The reactivity ofthe Caco-2 selected libraries L3.6B and L8.15B at cycle 5 (.33.3% and42.3%, respectively) as remarkably similar to that of colon A selectedphage (46.0%).

Table 5: Hybridization of Phage Populations With Oligonucleotide ELN93

[0087] Table 5.a: Caco2 Screening Cycles 1-6, Colon Screening Cycles 1-6& T-84 Screening Cycles 1-6

[0088] Table 5.b: Unselected Libraries L3.6, L3.15 & L8.15 TABLE 5.aCaco2 Caco2 Caco2 Caco2 Caco2 Caco2 Colon Colon T-84 T-84 T-84 T-84 T-84T-84 Cycle 3.6A 3.6B 3.15A 3.15B 8.15A 8.15B A B 3.6A 3.6B 3.15A 3.15B8.15A 8.15B 1 0 0.3 0 0 0 0 0.4 NA NA NA NA NA NA NA 2 0 0.3 0 0.4 0.31.0 4.7 NA 0 0 0 0 0 0 3 0 0 0 0.8 0 0 17.4 NA 0 0 0 0 0 0 4 0 0 0 1.20.3 0 26.4 NA 0 0 0 0 0 0 5 0 0 0 7.2 0 4.9 >20.0 NA NA NA NA NA NA NA 6NA NA NA NA NA NA 62.5¹ 0.6 0 0 0 0 0 0 (0.8)²

[0089] TABLE 5.b Unselected libraries ELN93 L3.6 0.8 L3.15 0.8 L8.15 0.3

Table 6: Hybridization of Phage Populations With Oligonucleotide ELN94

[0090] Table 6.a: Caco-2 Screening Cycles 1-6, Colon Screening Cycles1-6 & T84 Screening Cycles 1-6

[0091] Table 6.b: Unselected Libraries 13.6, L3.15 & L8.15 TABLE 6.aCaco2 Caco2 Caco2 Caco2 Caco2 Caco2 Colon Colon T-84 T-84 T-84 T-84 T-84T-84 Cycle L3.6A L3.6B 3.15A 3.15B 8.15A 8.15B A B 3.6A 3.6B 3.15A 3.15B8.15A 8.15B 1 0 0 0 0 0 0 0.4 NA NA NA NA NA NA NA 2 0 0.3 0 0 0 0 0 NA0 0 0 0.4 0 0.4 3 0 0 0 0 0 1.4 0 NA 0 0 2.8 0 0 2.8 4 0 6.0 0 1.6 0(3.8) 12.9 1.2 NA 4.0 0 12.8 4.4 0 0.4 5 3.3 >33.3 3.3 6.0 4.042.3 >40.0 NA NA NA NA NA NA NA 6 NA NA NA NA NA NA 46.0 26.2 0.4 0 0 00 0

[0092] TABLE 6.b Unselected libraries ELN94 L3.6 0 L3.15 0 L8.15 0

EXAMPLE 5 Identification of Peptide Sequences From Transported PhageAcross Caco-2 Tissue Samples

[0093] Caco-2 snapwells were prepared as described above and the ×30library was screened using Caco-2 cells according to the proceduresgiven above. FIG. 1 summarizes phage yield (% phage transported from theapical to basolateral medium) at cycles 1, 2, 3 and 4 in the basolateralmedium of polarized Caco-2 cells grown on snapwells. At each cycle thebasolateral medium was sampled both 1 hour and 24 hour post addition ofphage to the apical medium. Thus, following addition of the initialphage library at cycle one, the basolateral medium was removed after onehour and replaced with fresh basolateral medium. Subsequently, thebasolateral medium was removed 24 hours post addition of the initialphage library. In each case (one hour and 24 hour basolateral mediumsamples), the phage present was quantitated by titering a sample of eachbasolateral medium in Escherichia coli K91Kan strain. The remainingbasolateral medium from the one hour and twenty four hour sampling timepoint was combined, the phage present were PEG-precipitated, theprecipitated phage was resuspended in 100 μl of TBS and was used toinfect Escherichia coli K91Kan, thus permitting amplification of thephage present in the basolateral medium as outlined previously.Following amplification, the amplified phage was titered,PEG-precipitated, resuspended in TBS and titered. The phage suspensionwas now ready for the next round of further selection in the culturedCaco-2 cells, by repeating the steps above using the phage transportedinto the basolateral medium, as outlined previously. Upon going fromcycle 1 to 4, there was a 19.2 fold enrichment of phage which aretransported from the apical to basolateral medium of the Caco-2 cellsgrown on snapwells.

[0094] FIG. 2 summarizes the relative binding of 100 different phageisolates to fixed Caco-2 cells. The 100 individual phages from the ×30library were obtained from the cycle 4 selection (transport from apicalto the basolateral medium) of cultured Caco-2 cells grown on snapwells.For ELISA analysis, Caco-2 cells were grown to confluence in 96 welltissue culture plates as described above, followed by fixing in 10%formaldehyde as described above. The ELISA analysis was performed usingthe HRP-anti-M13 conjugate. In this figure, the binding of each phageisolate is arranged or presented so that the “weakest” to “strongest”binding phage are presented from left to right (and not the numericalnumber of the phage isolate). The binding of the negative control phage(M13mp18) and the absorbance readings obtained with untreated fixedCaco-2 cells is shown on the extreme right of FIG. 3, respectively.

[0095] FIG. 3 summarizes the binding of the top ten binders, clones 32,34, 39, 40, 53, 80, 84, 97, 98, and 100, to fixed Caco-2 cells, alongwith the binding of the negative control phage M13mp18 to the fixedCaco-2 cells, with phage binding monitored by ELISA analysis asdescribed above. The binding studies were performed in duplicate, usingneat phage (˜10¹⁰ pfu/ml) or diluted phage samples (diluted 1:25 and1:100 in each case). As a control, the absorbance readings obtainedusing the fixed Caco-2 cells in which no phage was added, is shown onthe right hand side of FIG. 3. FIG. 4 is essentially the same as FIG. 3,except that the background absorbance readings obtained using the fixedCaco-2 cells only, to which no phage was added, has been subtracted fromthe absorbance readings obtained using fixed Caco-2 cells which wereincubated with the indicated phage clone samples and the negativecontrol phage M13mp18. The precise titers of neat phage used for eachclone are given in Table 7. TABLE 7 TITERS OF NEAT PHAGE SAMPLES FOR THETOP TEN BINDERS CLONE pfu/ml 32 1.19 × 10¹⁰ 34 2.87 × 10¹⁰ 39 1.34 ×10¹⁰ 40 9.09 × 10⁹ 53 1.89 × 10¹⁰ 80 2.25 × 10¹⁰ 84 1.27 × 10¹⁰ 87 7.99× 10⁹ 98 1.99 × 10¹⁰ 100  8.36 × 10⁹

[0096] FIG. 5 is a graphical representation of the binding of the phageclones 39, 97 and 100, and the negative control phage M13mp18, to fixedCaco-2 cells using either neat phage samples (at ˜10¹⁰ pfu/ml) or thesame phage diluted 1:25 and 1:100. The phage binding experiments andsubsequent ELISA analysis was performed as previously outlined. Thisdata shows that the phage clones 39, 97 and 100 bind in a dose responsemanner, with reduction in the ELISA absorbance readings obtainedfollowing dilution of the phage either 1:25 or 1:100. In contrast, thenegative control phage M13mp18 does not bind in a dose response manner,with linear absorbance readings obtained using either neat, 1:25 or1:100 diluted phage.

[0097] The top ten binders, clones 32, 34, 39, 40, 53, 80, 84, 97, 98and 100 were sequenced using procedures outlined above. Eight of thesesequences were identical to the sequence of clone 97 giving DNA sequenceSEQ. NO. ID: 11 and peptide sequence SEQ. NO. ID: 12. The two remainingclones (53 and 100) produced individual isolates DNA SEQ. NOS. ID: 13and 15 with the corresponding peptide sequences SEQ. NOS. ID: 14 and 16,respectively. One skilled in the art could determine without undueexperimentation which fragments of these peptides permit or facilitatethe transport of an active agnet through a human or animal tissue. Onthe basis of the results of Example 4, it is expected that thesefragments consist of at least 6 amino acid residues.

EXAMPLE 6 Transport of Phage From Rat Lumen Into the Portal and SystemicCirculation

[0098] In this study, phage from random phage display libraries as wellas control phage were injected into the lumen of the ratgastrointestinal tract (in situ rat closed loop model). Blood wascollected over time from either the systemic circulation or portalcirculation and the number of phage which were transported to thecirculation was determined by titering blood samples in E. coli. At theconclusion of the collection of either systemic (300 min) or portalblood (60 min), the animals were sacrificed and brain, heart, kidney,spleen, ileum, duodenum, liver and pancreas tissue samples were removed,frozen in liquid nitrogen and stored at −80° C.

[0099] The phage display libraries used in this study were D38 and DC43in which gene III codes for random 38-mer and 43-mer peptides,respectively. As a negative control, the identical phage M13mp18, inwhich gene III does not code for a “random” peptide sequence, was used.Both the library phages D38 and DC43 were prepared from E. coli, mixedtogether, dialyzed against PBS, precipitated using PEG/NaCl and wereresuspended in PBS buffer. The M13mp18 control was processed in asimilar manner. The titer of each phage sample was determined and thephage samples were diluted in PBS to approximately the same titers priorto injection into the rat closed loop model.

[0100] For sampling from the systemic circulation, approximately IS cmof the duodenum of Wistar rats was tied off (closed loop model),approximately 0.5 ml of phage solution was injected into the closed loopand blood (0.4 ml) was sampled from the tail vein at various times. Thetime points used (in min) were: 0, 15, 30, 45, 60, 90, 120, 180, 240 and300 minutes. For sampling from the portal circulation, the portal veinwas catheterized, approximately 15 cm of the duodenum was tied off(closed loop model), 0.5 ml of phage solution was injected into theclosed loop and blood was sampled from the portal vein catheter atvarious times. As the portal sampling is delicate, sampling times wererestricted to 15, 30, 45 and 60 minutes, where possible. The volume ofphage injected into each animal was as follows: ANIMALS (15) VOLUME OFPHAGE INJECTED R1-R3 0.50 ml R4 0.43 ml R5-R15 0.45 ml

[0101] The estimated number of transported phage has been adjusted toaccount for differences in volume injected into each animal (using 0.5ml as the standard volume).

[0102] To investigate transport into the systemic circulation, animalsR1, R2 and R3 received the control phage M13mp18 and animals R4, R5, R6and R7 received the test phage D38/DC43 mix. To investigate transportinto the portal circulation, animals R8, R9 and R10 received the controlphage M13mp18 and animals R11, R12, R13 and R14 received the test phageD38/DC43 mix. Animal R15* received the combined phage samples fromanimals R4-R7 (see Table 8) which were sampled from the systemiccirculation on day one, followed by amplifiction in E. coli, PEGprecipitation and resuspension in PBS. On subsequent analysis, the titerof this phage was found to 100 times greater than the other phagesamples used for animals R8-R14. Thus, the date presented for animal R15in Table 9 is adjusted down.

[0103] Approximately 0.4 ml of the blood was collected at each timepoint in each model system. 30)l of the collected blood (systemic) wasmixed with 100 μl of the prepared E. coli strain K91Kan, incubated at37° C. for 30 min, and plated out for plaque formation using Top Agaroseon LB plates. Various negative controls were included in the titeringexperiments. The following day the number of plaques forming units(pfu's) was determined. Similarly, 30 μl of the collected blood (portal)and serial dilutions (1:100, 1:1000) thereof was mixed with 100 μl ofthe prepared E. coli strain K91Kan, incubated at 37° C. for 30 min, andplated out for plaque formation using Top Agarose on LB plates. Thefollowing day the number of plaques forming units (pfu's) wasdetermined.

[0104] In addition, approximately 300 μl of the collected blood fromeach time point (systemic and portal) was incubated with 5 ml ofprepared E. coli strain K91Kan in modified growth media containing 5 mMMgCl₂/MgSO4, incubated at 37° C. overnight with shaking (to permit phageamplification). The samples were centrifuged and the cell pellet wasdiscarded. Samples of the phage supernatant were collected seriallydiluted (10⁻², 10⁻⁴, 10⁻⁶, 10⁻⁸) in TBS buffer and were plated forplaques in order to determine the number of pfu's present in theamplified phage samples.

[0105] Furthermore, an aliquot of phage was removed from the “amplified”supernatants obtained from test animals #R4-R7 (samples from each timepoint were used), combined and was PEG-precipitated for two hours. Theprecipitated phage was resuspended in PBS buffer and was injected intoclosed loop model of animal #R15, followed by portal sampling.

[0106] The number of phage transported from the closed loop model intothe systemic circulation is presented in Table 8. The number of phagetransported from the closed loop model into the portal circulation ispresented in Table 9. These numbers are corrected for phage inputdifference and for volume input differences. Clearly, more phage arepresent in the portal samples than in the systemic samples, indicativeof either hepatic or RES clearance and/or phage instability in thesystemic circulation. In addition, the uptake of phage from the GIT intothe portal circulation is quite rapid, with substantial number of phagesdetected within 15 minutes. The results from the portal samplingexperiments would also indicate that the kinetics of uptake of phagefrom the D38/DC43 libraries is quicker than that of the control phage.Thus, there may be preferential uptake of phage coding for randompeptide sequences from the GIT into the portal circulation. In the caseof animals R13, R14 and R15*, the % of the phage transported into thetitered blood sample within the limited time frame (30, 45 and 15 mins,respectively) is estimated as 0.13%, 1.1% and 0.013%, respectively.TABLE 8 NUMBER OF PHAGE TRANSPORTED FROM THE CLOSED LOOP MODEL INTO THESYSTEMIC CIRCULATION Time (min) R1 R2 R3 R4 R5 R6 R7  0 0 0 0 0 0 0 0 15 0 1 9 0 0 1 7  30 2 1 0 0 46 1 11  45 10 4 2 1 32 0 20  60 63 19 211 114 0 21  90 104 20 18 3 115 0 22 120 94 24 27 0 64 0 6 180 94 12 23 1413 0 0 240 14 1 20 0 36 0 0 300 1 1 4 2 0 0 0 Total number of 382 83124 8 820 2 87 transported phage

[0107] TABLE 9 NUMBER OF PHAGE TRANSPORTED FROM THE CLOSED LOOP MODELINTO THE PORTALCIRCULATION Time (min) R8 R9 R10 R11 R12 R13 R14 R15* 1515 6 3 1 19 231,000 1,000,000 20,000 30  1 5 26 — 0 60,000 272,000 — 45— 1 555 — 1 — 1,240,000 — 60 — — — — 420,000 — — —

[0108] These studies demonstrate that both the control phage and theD38/DC43 phages are transported over time from the lumen of the GIT intothe portal and systemic circulation, as demonstrated by titering thephage transported to the blood in E. coli. More phage are transportedfrom the test phage samples into the portal circulation than thecorresponding control phage sample. In addition, the kinetics oftransport of the test phage into the portal circulation does appear toexceed that of the .control phage. Phage from the D38/DC43 librarieswhich appeared in the systemic circulation of different animals (R4-R7)were pooled, amplified in E. coli, precipitated, and re-applied to thelumen of the GIT, followed by collection in the portal circulation andtitering in E. coli. These selected phage were also transported from thelumen of the GIT into the portal circulation. This in situ loop modelmay represent an attractive screening model in which to identify peptidesequences which facilitate transport of phage and particles from the GITinto the circulation.

[0109] The frozen brain tissues for all the animals were thawed, cutinto small pieces, resuspended in 5 ml of sterile PBS containing acocktail of protease inhibitors (Boehringer) and homogenized in anUltrathorex homogenizer. Serial dilutions (neat and 10⁻², 10⁴, 10⁻⁶dilutions) were titered in E. coli. K91Kan starved bacteria. Inaddition, aliquots (100 μl) of the tissue homogenate were added to 100μl of E. coli K91Kan starved bacteria, incubated at 37° C. for 10 min,followed by addition of 5 ml of LB medium and incubation overnight at37° C. in a rotating incubator. Serial dilutions of amplified phage werethen titered in E. coli. No phage was detected in tissue homogenatesamples obtained from animals R4-R7 (sacrificed after systemic sampling)prior to amplification but phage were detected after amplification.Phage were detected prior to amplification in tissue homogenate samplesobtained from animals R11-R14.

[0110] An equal amount of phage from animals R11 through R14 were pooledat a concentration of 5×10¹¹ pfu/ml (in PBS). These phage were amplifiedas above, plated and the recombinant phage were picked and purified.Sequencing templates were prepared using the QIAprep Spin M13 columnsand were sequenced using the primer SEQ. NO. ID: 17 and SequenaseVersion 2.0 DNA sequencing kit. Eight DNA sequences (SEQ. NOS. ID:18,20,22,24,26,28,30, 32) with corresponding peptide sequences SEQ. NOS.ID: 19, 21, 23,25,27, 29, 31 and 33 were discovered. These peptidesequences, which were isolated because of their ability to transportphage (particles) from the GI tract to the brain, are capable offacilitating the transport of an active agent, such as a micro- ornanoencapsulated active agent, through a human or animal tissue.Additionally, one skilled in the art could determine without undueexperimentation which fragments or analogs of these peptides permit orfacilitate the transport of an active agnet through a human or animaltissue. On the basis of the results of Example 4, it is expected thatthese fragments consist of at least 6 amino acids.

[0111] Using this screening model system, a number of preselected phagelibraries now exist. These are the one pass brain tissue phage library,the one pass systemic phage library from animals R4-R7, a one-passportal library from animals R11-R14 and the two pass, rapid transport,systemic-portal phage library SP-2 from animal R15*.

EXAMPLE 7 Transport of Phage From Preselected Phage Libraries From theRat Lumen Into the Portal and Systemic Circulation

[0112] Four preselected phage libraries, GI-D, GI-S, GI-H and GI-P, areconstructed by pooling phage previously selected by screening randomphage display libraries D38 and DC43 using four distinct receptor orbinding sites located in the GIT. Similar to Example 7 above, thesepreselected phage libraries together with the negative control phageM13mp18 are injected into the rat closed loop model (6 animals perpreselected phage library), blood is collected over time from the portalcirculation via the portal vein and, at the termination of theexperiment, a systemic blood sample is collected from the tail vein andthe intestinal tissue region from the closed loop is collected.

[0113] In particular, phages selected in vitro to each receptor orbinding site located in the GIT were amplified in E. coli,PEG-precipitated, resuspended in TBS and the titer of each phage samplewas determined by plaquing in E. coli as described above. Subsequently,an equal number of each phage (8×10⁸ phage) for each receptor site waspooled into a preselected phage library together with the negativecontrol phage M13mp18 and each preselected phage library wasadministered to 6 Wistar rats per library (rats 1-6; GI-D, rats 7-12;GI-S, rats 13-18; GI-P, and rats 19-24; GI-H). Using the in situ loopmodel described above, 0.5 ml of preselected phage library solution wasinjected into the tied-off portion of the duodenum/jejunum. Blood wascollected into heparinised tubes from the portal vein at 0, 15, 30, 45and 60 minutes. A blood sample was taken from systemic circulation atthe end of the experiment. Similarly, the portion of theduodenum/jejunum used for phage injection was taken at the end of theexperiment.

[0114] 30 μl of the collected portal blood (neat and 10⁻², 10⁻⁴, 10⁻⁶dilutions) was added to 30 μl E. coli K91Kan cells (overnight cultureand incubated at 37° C. for 10 min. Subsequently, 3 ml of top agarosewas added and the samples were plated for plaques. 100 μl of thecollected portal blood was added to 100 μl of E. coli K91Kan. 5 ml of LBmedium was then added and the samples were incubated at 37° C. overnightin a rotating microbial incubator. The E. coli was removed bycentrifugation and the amplified phage supernatant samples were eithertitered directly or were PEG-precipitated, resuspended in TBS andtitered. Following titration of the amplified phage, samples containingphage from each set of animals were combined, adjusting the titer ofeach sample to the same titer, and were plated for plaques on LB agarplates (22 cm² square plates). Either 12,000 or 24,000 phage were platedfor plaques.

[0115] 30 μl of the collected systemic blood (neat and 10⁻², 10⁻⁴, 10⁻⁶dilutions) was added to E. coli K91Kan cells, incubated at 37° C. for 10min. Three ml of top agarose was then added and the samples were platedfor plaques. 100 μl of the collected systemic blood was added to 100 μlof E. coli K91Kan, incubated at 37° C. for 10 min. 5 ml of LB medium wasthen added and the samples were incubated at 37° C. overnight in arotating microbial incubator. The E. coli was removed by centrifugationand the amplified phage supernatant samples were either titered directlyor were PEG-precipitated, resuspended in TBS and titered. Followingtitration of the amplified phage, samples containing phage from each setof animals were combined, adjusting the titer of each sample to the sametiter, and were plated for plaques on LB agar plates (22 cm² squareplates). Either 12,000 or 24,000 phage were plated for plaques.

[0116] The intestinal tissue portion used in each closed loop wasexcised. The tissue was cut into small segments, followed by 3 washingsin sterile PBS containing protease inhibitors, and homogenized in anUltra thorex homogeniser (Int-D samples). Alternatively, the tissue (inPBS supplemented with protease inhibitors) was homogenized in an UltraThorex homogeniser, washed 3 times in PBS containing protease inhibitorsand resuspended in PBS containing protease inhibitors (Int-G samples).In each case, serial dilutions (neat and 10⁻², 10⁻⁴, 10⁻⁶ dilutions) ofthe tissue homogenate was titered in E. coli. In addition, an aliquot(100 μl) of the tissue homogenate was added to 100 μl of E. coli K91Kan,incubated at 37° C. for 10 min. followed by addition of 5 ml of LBmedium and incubation overnight at 37° C. in a rotating microbialincubator.

[0117] The phage amplified from the portal blood, systemic blood andintestinal tissue was plated for plaques. The plaques were transferredto Hybond-N Nylon filters, followed by denaturation (1.5M NaCl, 0.5MNaOH), neutralization (0.5M TRIS-HCl, pH 7.4, 1.5M NaCl), washing in2×SSC buffer. The filters were air-dried, and the DNA was cross-linkedto the filter (UV crosslinking: 2 min, high setting). The filters wereincubated in pre-hybridization buffer (6×SSC, 5× Denhardt's solution,0.1% SDS, 20 μg/ml yeast tRNA) at 40° C.-45° C. for at least 60 min.

[0118] Synthetic oligonucleotides, (22-mers), complimentary to regionscoding for the receptor or binding sites used to create the preselectedphage library, were synthesized. The oligonucleotides (5 pmol) were 5′end labelled with ³²P-ATP and T4 polynucleotide kinase and approximately2.5 pmol of labelled oligonucleotide was used in hybridization studies.Hybridization's were performed at 40-45° C. overnight in buffercontaining 6×SSC, 5× Denhardt's solution, 0.1% SDS, 20 μg/ml yeast tRNAand the radiolabeled synthetic oligonucleotide, followed by washings(20-30 min at 40-45° C.) in the following buffers: (i) 2×SSC 0.1% SDS,(ii) 1×SSC/0.1% is SDS, (iii) 0.1×SSC/0.1% SDS. The filters wereair-dried and exposed for autoradiography for 15 hours, 24 hours or 72hours.

[0119] Table 10 summarises the results from the hybridization studiesoutlined above. Apart from the synthetic oligonucleotide to HAX9, alloligonucleotides were initially confirmed to be radiolabeled, asdetermined by hybridisation to the corresponding phage target (eg.,phage S15 hybridised to the oligonucleotide S15). In addition, under theexperimental conditions used the oligonucleotides essentially did nothybridise to the negative control phage template M13mp18. Twooligonucleotides were synthesised to the phage M13mp18—(1) a positiveoligonucleotide which hybridises to a conserved sequence in both M13mp18and each of the GIT receptor or GIT binding site selected phages[designated M13(positive)] in Table 10 and (2) a negativeoligonucleotide which only hybridises to a sequence unique to themultiple cloning site of phage M13mp18 and which does not hybridise toany of the GIT receptor or GIT binding site selected phages. TABLE 10SUMMARY OF HYBRIDIZATION RESULTS A: (GI-S) Phage Portal Int.-G Int.-DS15 ++ +/− +/− S21 − − − S22 − −/+ − SNI-10 +++/+ ++ ++ SNI-28 − − −SNI-34 ++ − − SNI-38 ++ − − SNI-45 − − − SNIAX-2 − − − SNIAX-6- − −SNIAX-8 − − − M13 (positive) ++++++ ++++++ ++++++ M13 (negative) ND + −B: (GI-D) Phage Portal Int.-G Int.-D DAB3 +++ +/− −/+ DAB7 ++ ++ −/+DAB10 ++++++ +/− −/+ DAB18 − − − DAB24 − − − DAB30 ++++ ++ +++ DAX15 − −− DAX23 −/+ + −/+ DAX24 − − − DAX27 − + − DCX8 +++++ +/− − DCX11 ++++++++ −/+ DCX26 − − − DCX33 +++ ++ ++ DCX36 − − − DCX39 − −/+ − DCX42 − −−/+ DCX45 − ++ − M13 (positive) ++++++ ++++++ ++++++ M13 (negative) +/−−/+ − C: (GI-H) Phage Int-G Portal Systemic H40 − − ++++ HAX9 ND ND NDHAX35 − + − HAX40 − − − HAX42 − ++ ++ HCA3 − − − PAX2 − +++ ++++ M13(positive) ++++++ ++++++ ++++++ M13 (negative) − −−/+ − D: (GI-P) PhageInt-G Portal Systemic PAX2 − ++ − PAX9 ++ +++ − PAX14 − ++ − PAX15 −/+ −− PAX16 − − − PAX17 + ++/+ − PAX18 − − − PAX35 − − − PAX38 −/+ − −PAX40 + +++ − PAX43 + − − PAX45 − − − PAX46 − +++ − P31 ++ ++++ ++ P90 −− − 5PAX3 ++/+ ++ − 5PAX5 − − ++ 5PAX7 +++ − − 5PAX12 ++++ ++ − H40 ++++ − M13 (positive) ++++++ ++++++ ++++++ M13 (negative) − − −

[0120] In the case of the GI-S pool of phages, only four phages aretransported from the closed loop model into the portalcirculation—phages S15, SNI-10, SNI-34 and SNI-38. The other phages,S21, S22, SNI-28, SNI45, SNIAX-2, SNIAX-6 and SNIAX-8 are nottransported from the GIT into the portal circulation. In addition,phages SNI-10 and to a lesser extent phages S15 and S22 were found inthe intestine samples or fractions, whereas the other phages were not.There was a very low presence (<0.1%) of the phage M13mp18 in the Int-Gsamples. These results show that phages can be further selected frompre-selected libraries, permitting the identification of phages whichare transported from the GIT closed loop into the portal circulation orphages which bind to or are internalised by intestinal tissue.

[0121] In the case of the GI-D pool of phages, there is a rank order bywhich phages are transported from the GIT closed loop model into theportal circulation, with phages DCX11 and DAB 10 preferably transported,followed by phages DCX8, DAB30, DAB3 and DAB7. A number of phages fromthis pool are not transported into the portal circulation, includingphages DAB18, DAB24, DAX15, DAX24, DAX27, DCX26, DCX36, DCX39, DCX42,DCX45. There is a very low level of transport of phage DAX23 from theGIT into the portal circulation. Similarly, only some of the phages arefound in the intestinal samples fractions, including phages DAB30,DCX33, DAB7, DCX11, DCX45 and to a much lesser extent phages DAB3,DAB10, DCX8, DCX39, DCX42. Some phages are not found in the intestinalsamples, including phages DAB18, DAB24, DAX15, DAX24, DCX26, and DCX36.There was a very low presence (<0.1%) of the phage M13mp18 in the Int-Gsamples. These results show that phages can be further selected frompre-selected libraries, permitting the identification of phages whichare transported from the GIT closed loop into the portal circulation orphages which bind to or are internalised by intestinal tissue.

[0122] In the case of the GI-H pool of phages, there is a rank order bywhich phages are transported from the GIT closed loop model into theportal or systemic circulation, with phages PAX2 (which was used at a 4×concentration relative to the other phages in this pool) followed byphage HAX42 found in the portal and systemic circulation and phage H40found in the systemic circulation only. None of the phages in this poolwere found in the intestine samples or fractions. Phage M13mp18 was notfound in the intestine fractions or systemic circulation, with very lowincidence (<0.001%) in the portal circulation. These results show thatphages can be further selected from pre-selected libraries, permittingthe identification of phages which are transported from the GIT closedloop into the portal and/or systemic circulation or phages which bind toor are internalised by intestinal tissue.

[0123] In the case of the GI-P pool of phages, the phages PAX2 and H40were also included in this pool. A number of phages from this pool werefound in the portal circulation, including phages P31, PAX46, PAX9, H40,PAX17, PAX40, PAX2, PAX14, 5PAX3 and 5PAX12. A number of phages were notfound in the portal blood including the negative control phage M13mp18,PAX15, PAX16, PAX18, PAX35, PAX38, PAX43, PAX45, P90, 5PAX5 and 5PAX7.The only phage found in the systemic circulation were phages 5PAX5 andP31. In addition, there was preferential binding of some phages to theintestine, including phages 5PAX12, 5PAX7, 5PAX3, H40, P31, PAX9, and toa lesser extent phages PAX38 and PAX5. Some phages were not found in theintestine samples, including the negative control phage M13mp18 and thephages PAX2, PAX14, PAX16, PAX18, PAX3S, PAX45, PAX46, P90 and 5PAX5.These results show that phages can be further selected from pre-selectedlibraries, permitting the identification of phages which are transportedfrom the GIT closed loop into the portal and/or systemic circulation orphages which bind to or are internalised by intestinal tissue.

[0124] The present invention is not to be limited in scope by thespecific embodiments described herein. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

1 33 19 base pairs nucleic acid single linear other nucleic acid /desc =“gene VIII primer ELN 71” 1 AGTAGCAGAA GCCTGAAGA 19 179 base pairsnucleic acid single linear other nucleic acid /desc = “Class of 9Clones” 2 AAGCTTTGCC GTTTGCGCCT TGTGGTTATA AGCATCCTAC TTGTCGTGTGGAGCCTGCAG 60 ACGCCACATA ATAAACAGCG GCGCAGTATA ACCCCAAGGC GGAATGCTGCAGGGACGTTG 120 GCAAAGCTTT CCGGTTTCGG CTCGGATTTA TTATGGGTAT GCATGATTCTCCTGATCCT 179 162 base pairs nucleic acid single linear other nucleicacid /desc = “Class of 5 clone” 3 AAGCTTTGCC CTTACTAGCA GATGCCTGAGCTGTATTCTC CTCATCGTTT TTGTCCTGCA 60 GATATACGCC ATATACAGCG GATAAGTAAAAATAGTAGGA GTAAGCAAAG CTTTGCCCTC 120 GTCAGCTGTA TCCTGCGCCG CCGACTGAGCTTACTGTGCG TC 162 170 base pairs nucleic acid single linear othernucleic acid /desc = “Class of 3 clones” 4 AAGCTTTGCC TGCGTAGGCCTATTCCTTCT TTGCTGCCGG TGCCACTTGT ATCGCTGCAG 60 GCTTAGTATA GAGGCCCAAAAATAGGAGAA GGCACCAGAT ATAGATGCAG GACGTTGGCA 120 AACTTTGCGG CTGTCTAACCGATTGTTCGG CCTCTGCATT TGTACTGGTC 170 158 base pairs nucleic acid singlelinear other nucleic acid /desc = “Individual isolate” 5 TCGCATTCTCCGGGCCTTTT GCGAATTTTC GGCAATGGTT GCGTCCTGCA GGAAACCCAA 60 ACGCCCACAAACACGCAGAA GACGCCGGAG AAAAAGTGCA AAGCTTTGCC ATTTTGCTGC 120 CTAGGATTCCGCATCCGTTT GTGTCCGGCT CCTTTGTC 158 163 base pairs nucleic acid singlelinear other nucleic acid /desc = “individual isolate” 6 GGGCTTCGGGTTACTTGTAC TCGGCGTCCT CCTCTTTGTC CTGCAGGCAA ATAAGGCTGC 60 TGACACCTAGTAGTGCGAAG ACAGCCTCTG CAGGGAAGTT GGCAAAGCTT TGCCGGGCCG 120 ATTTCAGGTGTTCCTCTTGA TGTTTTTTGC TTTTTGGGTT GTC 163 141 base pairs nucleic acidsingle linear other nucleic acid /desc = “individual isolate” 7CTCAGCCTTA TTAGGTGCCT TGGATCCTCT GGTCGCCTAT GCCTGCAGCA AGCAGTAGTA 60TACCATAGTA GAGGCAGAGT CTACATGCAA AGCTTTGCCT CCTATGTTAG AGTCCGGATA 120GTGGGTCTTT GTCGGAGTCC C 141 129 base pairs nucleic acid single linearother nucleic acid /desc = “individual isolate” 8 TTGGTTGGTT ATCGGCTCAGCGTCTCTGCA GCCGCAAGTC GAAACGCGAC CACGAAGTCA 60 GATACTCCAA AAAGCAAAGCTTTGCCTGTC AGTCGCCTAG GTAGCGTGCT TCTCGGTCTC 120 TGCGGCCTC 129 126 basepairs nucleic acid single linear other nucleic acid /desc = “individualisolate” 9 TCTACGCAGT TCCGTTGTGG GTGTCTTGTT CCTCCTACTC CTCCTGCAGGAAAAGAACAC 60 TCCAGCACGA TGAGGAATCT CCTAAAAAAT AGTCTGCAGG AGTTGCAAAGCTTTGCCTTG 120 TTGCCG 126 45 base pairs nucleic acid single linear othernucleic acid /desc = “individual isolate” 10 GTTAGACGGT GCAGGCGCCTATTAATCAGC CTGAGGATTG GCCTC 45 118 base pairs nucleic acid single linearDNA (genomic) CDS 2..103 11 T GCA GAT GAT TTT ATG CAG TGC ATG CTA ACTTTG CCA ACG TCC CTG 46 Ala Asp Asp Phe Met Gln Cys Met Leu Thr Leu ProThr Ser Leu 1 5 10 15 CAG CAG GAG CAG TCT CCC TAT AAT TAC TAC GAC ACCCAT GAA GCG AAT 94 Gln Gln Glu Gln Ser Pro Tyr Asn Tyr Tyr Asp Thr HisGlu Ala Asn 20 25 30 CAA CCT CAC GCTGCAGAAG GTGAT 118 Gln Pro His 34amino acids amino acid linear protein 12 Ala Asp Asp Phe Met Gln Cys MetLeu Thr Leu Pro Thr Ser Leu Gln 1 5 10 15 Gln Glu Gln Ser Pro Tyr AsnTyr Tyr Asp Thr His Glu Ala Asn Gln 20 25 30 Pro His 120 base pairsnucleic acid single linear DNA (genomic) CDS 16..105 13 ATGCTATCGT TTGCCACG CCG ACG ACG ACC GCT ACC GTA GTA GGG ACG ACT 51 Thr Pro Thr Thr ThrAla Thr Val Val Gly Thr Thr 35 40 45 CAG CCT GTT GAT TTG TCT AGT AAG CATCTG CTT AGG CAT CCT TGT CGT 99 Gln Pro Val Asp Leu Ser Ser Lys His LeuLeu Arg His Pro Cys Arg 50 55 60 GAG TTT GCTGCAGAAG GTGAT 120 Glu Phe 30amino acids amino acid linear protein 14 Thr Pro Thr Thr Thr Ala Thr ValVal Gly Thr Thr Gln Pro Val Asp 1 5 10 15 Leu Ser Ser Lys His Leu LeuArg His Pro Cys Arg Glu Phe 20 25 30 120 base pairs nucleic acid singlelinear DNA (genomic) CDS 16..105 - 31..33 /product= “GLN” /label= GLN/note= “Amber suppressor SupE in E. coli strain K91Kan reads in-framestop codons TAG as GLN residues” - 43..45 /product= “GLN” /label= GLN/note= “Amber suppressor SupE in E.coli strain K91Kan reads in-framestop codon TAG as GLN residues” 15 ATGCTATCGT TTGCC ATG TCG CCT GAT CATTAG TAT GCG CTT TAG TCG TCC 51 Met Ser Pro Asp His Gln Tyr Ala Leu GlnSer Ser 35 40 TTT GTC TTG CCG TGT TGT CGG CCT CTT CTG GTT GAT TCT GATTAT ATT 99 Phe Val Leu Pro Cys Cys Arg Pro Leu Leu Val Asp Ser Asp TyrIle 45 50 55 CAT TCT GCTGCAGAAG GTGAT 120 His Ser 60 30 amino acidsamino acid linear protein 16 Met Ser Pro Asp His Gln Tyr Ala Leu Gln SerSer Phe Val Leu Pro 1 5 10 15 Cys Cys Arg Pro Leu Leu Val Asp Ser AspTyr Ile His Ser 20 25 30 20 base pairs nucleic acid single linear othernucleic acid /desc = “”geneIII primer ELN77a“” 17 CCCTCATAGT TAGCGTAACG20 109 base pairs nucleic acid single linear DNA (genomic) CDS 5..109 18CTCG AGG GGG TAC GGC CGG TTG GCG GAG TCG TGT TGT GTG AAC GAT CGT 49 ArgGly Tyr Gly Arg Leu Ala Glu Ser Cys Cys Val Asn Asp Arg 35 40 45 TGT ATTCGT ACC GTC GGG GGT TGT GGT AAT TCC CCT GCC TCC GAC ATC 97 Cys Ile ArgThr Val Gly Gly Cys Gly Asn Ser Pro Ala Ser Asp Ile 50 55 60 CTC TCC AACACG 109 Leu Ser Asn Thr 65 35 amino acids amino acid linear protein 19Arg Gly Tyr Gly Arg Leu Ala Glu Ser Cys Cys Val Asn Asp Arg Cys 1 5 1015 Ile Arg Thr Val Gly Gly Cys Gly Asn Ser Pro Ala Ser Asp Ile Leu 20 2530 Ser Asn Thr 35 124 base pairs nucleic acid single linear DNA(genomic) CDS 5..118 20 CTCG AGC ACC CCA GGT CGT GGC TCC GGT CGG GAT ACGGGG GCC AAC AAC 49 Ser Thr Pro Gly Arg Gly Ser Gly Arg Asp Thr Gly AlaAsn Asn 40 45 50 GCG GCT GAC ACC CCT TAC GCC AAT CCC TCT CAC CGC GAC ACGATC CTT 97 Ala Ala Asp Thr Pro Tyr Ala Asn Pro Ser His Arg Asp Thr IleLeu 55 60 65 TCC CTC GAC CCC TCC CTT CTC TCTAGA 124 Ser Leu Asp Pro SerLeu Leu 70 38 amino acids amino acid linear protein 21 Ser Thr Pro GlyArg Gly Ser Gly Arg Asp Thr Gly Ala Asn Asn Ala 1 5 10 15 Ala Asp ThrPro Tyr Ala Asn Pro Ser His Arg Asp Thr Ile Leu Ser 20 25 30 Leu Asp ProSer Leu Leu 35 127 base pairs nucleic acid single linear DNA (genomic)CDS 5..121 22 CTCG AGG CAG CAC CTC GTC GTT CGT GAC TTG CAT GAG CCT CGTTTC CGC 49 Arg Gln His Leu Val Val Arg Asp Leu His Glu Pro Arg Phe Arg40 45 50 GAC ACT AAT ACC GGT GTC CAC GCC ACG TTC TCG CCG CCT GTC TCC GTC97 Asp Thr Asn Thr Gly Val His Ala Thr Phe Ser Pro Pro Val Ser Val 55 6065 GCT ACC GAC CAC CGC ACC CCG CCC TCTAGA 127 Ala Thr Asp His Arg ThrPro Pro 70 75 39 amino acids amino acid linear protein 23 Arg Gln HisLeu Val Val Arg Asp Leu His Glu Pro Arg Phe Arg Asp 1 5 10 15 Thr AsnThr Gly Val His Ala Thr Phe Ser Pro Pro Val Ser Val Ala 20 25 30 Thr AspHis Arg Thr Pro Pro 35 127 base pairs nucleic acid single linear DNA(genomic) CDS 5..121 24 CTCG AGT TTC AGC AAC CTT ACC GCC GGT GAT GAG GAGGAT GAT CAC TTC 49 Ser Phe Ser Asn Leu Thr Ala Gly Asp Glu Glu Asp AspHis Phe 40 45 50 TCG GGT GGG CGG TTC AAT CAC GCC AAT CTT ACT AGC CGG TCCCAT AAT 97 Ser Gly Gly Arg Phe Asn His Ala Asn Leu Thr Ser Arg Ser HisAsn 55 60 65 70 CGT GGG CAG CTG GCT AGT TCC GCC TCTAGA 127 Arg Gly GlnLeu Ala Ser Ser Ala 75 39 amino acids amino acid linear protein 25 SerPhe Ser Asn Leu Thr Ala Gly Asp Glu Glu Asp Asp His Phe Ser 1 5 10 15Gly Gly Arg Phe Asn His Ala Asn Leu Thr Ser Arg Ser His Asn Arg 20 25 30Gly Gln Leu Ala Ser Ser Ala 35 127 base pairs nucleic acid single linearDNA (genomic) CDS 5..121 26 CTCG AGG CAG AGT GTC TTG GAC AGC TGG GGG GGTAAG ACG AGT GTT ACG 49 Arg Gln Ser Val Leu Asp Ser Trp Gly Gly Lys ThrSer Val Thr 40 45 50 GGG AGC CTG GAG CGC TAT TAC GCC AGC CAC TCT CAC ACTAGT GCC CCC 97 Gly Ser Leu Glu Arg Tyr Tyr Ala Ser His Ser His Thr SerAla Pro 55 60 65 70 ACT CCC CAC TAC GCC TCC CAC TCT TCTAGA 127 Thr ProHis Tyr Ala Ser His Ser 75 39 amino acids amino acid linear protein 27Arg Gln Ser Val Leu Asp Ser Trp Gly Gly Lys Thr Ser Val Thr Gly 1 5 1015 Ser Leu Glu Arg Tyr Tyr Ala Ser His Ser His Thr Ser Ala Pro Thr 20 2530 Pro His Tyr Ala Ser His Ser 35 127 base pairs nucleic acid singlelinear DNA (genomic) CDS 5..121 28 CTCG AGA CAG TGG GTG GGT GAC CGG GCGGAT GGG GAG GGG AAC TGG GTT 49 Arg Gln Trp Val Gly Asp Arg Ala Asp GlyGlu Gly Asn Trp Val 40 45 50 GAC GAG AAG TAT AGT CGG GAC GCC AAT GTC ATTTCG TAC CGG AAG CAC 97 Asp Glu Lys Tyr Ser Arg Asp Ala Asn Val Ile SerTyr Arg Lys His 55 60 65 70 AAC CAT GCG AGC CAG GGC ACC CTC TCTAGA 127Asn His Ala Ser Gln Gly Thr Leu 75 39 amino acids amino acid linearprotein 29 Arg Gln Trp Val Gly Asp Arg Ala Asp Gly Glu Gly Asn Trp ValAsp 1 5 10 15 Glu Lys Tyr Ser Arg Asp Ala Asn Val Ile Ser Tyr Arg LysHis Asn 20 25 30 His Ala Ser Gln Gly Thr Leu 35 103 base pairs nucleicacid single linear DNA (genomic) CDS 5..103 30 CTCG AGG GCG AGT GAT TGTGAT GTC GAG TGT AAC CTG CGC TGG GTG GAG 49 Arg Ala Ser Asp Cys Asp ValGlu Cys Asn Leu Arg Trp Val Glu 40 45 50 GAT GTG GGG GGG GTG TGG TAC GCCAAG ACC GTT TCG CGA ATG CTA AGC 97 Asp Val Gly Gly Val Trp Tyr Ala LysThr Val Ser Arg Met Leu Ser 55 60 65 70 ACG ACG 103 Thr Thr 33 aminoacids amino acid linear protein 31 Arg Ala Ser Asp Cys Asp Val Glu CysAsn Leu Arg Trp Val Glu Asp 1 5 10 15 Val Gly Gly Val Trp Tyr Ala LysThr Val Ser Arg Met Leu Ser Thr 20 25 30 Thr 142 base pairs nucleic acidsingle linear DNA (genomic) CDS 5..136 32 CTCG AGA CAG TCT GCG GGC GTGTTG GGT TTT GCG CCT ACC AAT ATC GAT 49 Arg Gln Ser Ala Gly Val Leu GlyPhe Ala Pro Thr Asn Ile Asp 35 40 45 GAC ACT AGC TTT CAT GCG GGT TGT GGTGAC ACA TTG GCG ATT CCG TGC 97 Asp Thr Ser Phe His Ala Gly Cys Gly AspThr Leu Ala Ile Pro Cys 50 55 60 CGA CAT CGT TCC TCC CTG ATC AGC CCT GCTCGC CCT CCC TCTAGA 142 Arg His Arg Ser Ser Leu Ile Ser Pro Ala Arg ProPro 65 70 75 44 amino acids amino acid linear protein 33 Arg Gln Ser AlaGly Val Leu Gly Phe Ala Pro Thr Asn Ile Asp Asp 1 5 10 15 Thr Ser PheHis Ala Gly Cys Gly Asp Thr Leu Ala Ile Pro Cys Arg 20 25 30 His Arg SerSer Leu Ile Ser Pro Ala Arg Pro Pro 35 40

What is claimed is:
 1. A method of identifying a peptide which permitsor facilitates the transport of an active agent through a human oranimal tissue, comprising the steps of: (a) administering in vivo or insitu to a site in an animal a predetermined amount of phage from arandom phage library or a preselected phage library; (b) at apredetermined time harvesting phage from the animal to selecttransported phage, wherein the harvesting site is separated from thesite of administration by a tissue barrier, (c) amplifying thetransported phage in a host; (d) repeating in order step (a) using thetransported phage obtained in step (b) and amplified in step (c) andsteps (b) and (c) a predetermined number of times to obtain a selectedphage library containing phage which can be transported from the site ofadministration to the site of harvesting; and (e) determining theidentity of at least one peptide coded by phage in the selected phagelibrary to identify a peptide which permits or facilitates the transportof an active agent through a human or animal tissue.
 2. The method ofclaim 1, wherein step (d) is repeated from 0 to 30 times.
 3. The methodof claim 1, wherein the tissue barrier is selected from the groupconsisting of duodenum tissue, jejunum tissue, ilium tissue, ascendingcolon tissue, transverse colon tissue, desending colon tissue, pelviccolon tissue, tissue in the vascular endothelium which lines thevascular system, tissue in the vascular endothelium of the blood brainbarrier, vascular smooth muscle tissue, alveolar tissue, liver tissue,kidney tissue, bone marrow tissue, heart tissue, spleen tissue, pancreastissue, thymus tissue, brain tissue, spinal tissue, neuronal tissue,retinal eye tissue or combinations thereof.
 4. The method of claim 1,wherein the tissue barrier comprises epithelial cells lining the lumenalside of the gastrointestinal track.
 5. The method of claim 4, whereinthe tissue barrier comprises the colon tissue.
 6. The method of claim 1,wherein the phage is administered to the gastro-intestinal tract and isharvested from portal blood.
 7. The method of claim 1, wherein the phageis administered to the gasto-intestinal tract and is harvested fromsystemic blood.
 8. The method of claim 1, wherein the active agent is adrug or antigen.
 9. The method of claim 1, wherein the active agent is anano- or microparticle.
 10. The method of claim 9, wherein the peptideis coated onto or adsorbed onto or covalently bonded to the surface ofthe nano-or microparticle.
 11. The method of claim 9, wherein the nano-or microparticle is formed from the peptide.
 12. The method of any ofclaims 9, wherein the nano- or microparticle is a drug-loaded ordrug-encapsulated nano- or microparticle.
 13. A method of identifying apeptide which permits or facilitates the transport of an active agentthrough a human or animal tissue, comprising the steps of: (a)administering in vivo or in situ to the gastro-instestinal tract of ananimal a predetermined amount of phage from a random phage library or apreselected phage library; (b) at a predetermined time harvesting phagewhich is transported to the portal circulatory system or to the systemiccirculatory system of the animal to select transported phage; (c)amplifying the transported phage in a host; (d) repeating in order step(a) using the transported phage obtained in step (b) and amplified instep (c) and steps (b) and (c) a predetermined number of times to obtaina selected phage library containing phage which can be transported fromthe gastrointestinal tract to the portal circulatory system or to thesystemic circulatory system of the animal; and (e) determining theidentity of at least one peptide coded by phage in the selected phagelibrary to identify a peptide which permits or facilitates the transportof an active agent through a human or animal tissue.
 14. A peptide whichpermits or facilitates the transport of an active agent through a humanor animal tissue, the peptide being identified by a method comprisingthe steps of: (a) administering in vivo or in situ to a site in ananimal a predetermined amount of phage from a random phage library or apreselected phage library; (b) at a predetermined time harvesting phagefrom the animal to select transported phage, wherein the harvesting siteis separated from the site of administration by a tissue barrier, (c)amplifying the transported phage in a host; (d) repeating in order step(a) using the transported phage obtained in step (b) and amplified instep (c) and steps (b) and (c) a predetermined number of times to obtaina selected phage library containing phage which can be transported fromthe site of administration to the site of harvesting; and (e)determining the identity of at least one peptide coded by phage in theselected phage library to identify a peptide which permits orfacilitates the transport of an active agent through a human or animaltissue.
 15. The peptide of claim 14, wherein step (d) is repeated from 0to 30 times.
 16. The peptide of claim 14, wherein the tissue barriercomprises duodenum tissue, jejunum tissue, ilium tissue, ascending colontissue, transverse colon tissue, desending colon tissue, pelvic colontissue, tissue in the vascular endothelium which lines the vascularsystem, tissue in the vascular endothelium of the blood brain barrier,vascular smooth muscle tissue, alveolar tissue, liver tissue, kidneytissue, bone marrow tissue, heart tissue, spleen tissue, pancreastissue, thymus tissue, brain tissue, spinal tissue, neuronal tissue,retinal eye tissue or combinations thereof.
 17. The peptide of claim 14,wherein the tissue barrier comprises epithelial cells lining the lumenalside of the gastro-intestinal track.
 18. The peptide of claim 17,wherein the tissue barrier comprises colon tissue.
 19. The peptide ofclaim 14, wherein the phage is administered to the gastro-intestinaltract and is harvested from portal blood.
 20. The peptide of claim 14,wherein the phage is administered to the gastro-intestinal tract and isharvested from systemic blood.
 21. The peptide of claim 14, wherein theactive agent is a drug or antigen.
 22. The peptide of claim 14, whereinthe active agent is a nano- or microparticle.
 23. The peptide of claim22, wherein the peptide is coated onto or adsorbed onto or covalentlybonded to the surface of the nano-or microparticle.
 24. The peptide ofclaim 22, wherein the nano- or microparticle is formed from the peptide.25. The peptide of claim 22, wherein the nano- or microparticle is adrug-loaded or drug-encapsulated nano- or microparticle.
 26. A method ofidentifying a peptide motif which when present in a peptide permits orfacilitates the transport of an active agent through a human or animaltissue, comprising the steps of: (a) administering in vivo or in situ toa site in an animal a predetermined amount of phage from a random phagelibrary or a preselected phage library; (b) at a predetermined timeharvesting phage from the animal to select transported phage, whereinthe harvesting site is separated from the site of administration by atissue barrier, (c) amplifying the transported phage in a host; (d)repeating in order step (a) using the transported phage obtained in step(b) and amplified in step (c) and steps (b) and (c) a predeterminednumber of times to obtain a selected phage library containing phagewhich can be transported from the site of administration to the site ofharvesting; and (e) determining the identity of a plurality of peptidescoded by phage in the selected phage library to identify a peptide motifcommon to at least two of the peptides which permit or facilitate thetransport of an active agent through a human or animal tissue.
 27. Amethod of identifying a peptide which permits or facilitates theinternalization of an active agent into human or animal tissue,comprising the steps of: (a) administering in vivo or in situ to a siteadjacent a selected tissue in an animal a predetermined amount of phagefrom a random phage library or a preselected phage library; (b) at apredetermined time harvesting phage from the selected tissue to selecttissue internalized phage, wherein the harvesting comprising the stepsof: (i) homognizing the selected tissue to obtain a homogenate: and (ii)washing the homogenate; (c) amplifying the tissue internalized phage ina host; (d) determining the identity of at least one peptide coded bytissue internalized phage to identify a peptide which permits orfacilitates the internalization of an active agent into a human oranimal tissue or transport of the active agent through human or animaltissue.
 28. The method of claim 27, wherein the preselected phagelibrary comprises tissue internalized phage.
 29. The method of claim 27,wherein the selected tissue is selected from the group consisting ofduodenum, jejunum, ilium, ascending colon, transverse colon, desendingcolon, pelvic colon, vascular endothelium which lines the vascularsystem, vascular endothelium of the blood brain barrier, vascular smoothmuscle, alveolar, liver, kidney, bone marrow, heart, spleen, pancreas,thymus, brain, spinal cord, neuronal or retinal eye tissue.
 30. A methodof identifying a peptide which permits or facilitates the transport ofan active agent through a human or animal tissue, comprising the stepsof: (a) administering in vivo or in situ to a site adjacent a selectedtissue in an animal a predetermined amount of phage from a random phagelibrary or a preselected phage library; (b) at a predetermined timeharvesting phage from the selected tissue to obtain tightly bound phage,wherein the harvesting comprises the steps of: (i) washing the selectedtissue to obtain washed tissue; and (ii) homogenized the washed tissue(c) amplifying the tightly bound phage in a host; and (d) determiningthe identity of at least one peptide coded by tightly bound phage toidentify a peptide which permits or facilitates the internalization ofan active agent into a human or animal tissue.
 31. The method of claim30, wherein the preselected phage library comprises tightly bound phage.32. The method of claim 30, wherein the selected tissue is selected fromthe group consisting of duodenum, jejunum, ilium, ascending colon,transverse colon, desending colon, pelvic colon, vascular endotheliumwhich lines the vascular system, vascular endothelium of the blood brainbarrier, vascular smooth muscle, alveolar, liver, kidney, bone marrow,heart, spleen, pancreas, thymus, brain, spinal cord, neuronal or retinaleye tissue.
 33. The method of claim 1, wherein the phage is administeredto the gastrointestinal tract and is harvested from brain tissue. 34.The method of claim 1, wherein the havesting site is tissue selectedfrom the group consisting of duodenum, jejunum, ilium, ascending colon,transverse colon, desending colon, pelvic colon, vascular endotheliumwhich lines the vascular system, vascular endothelium of the blood brainbarrier, vascular smooth muscle, alveolar; liver, kidney, bone marrow,heart, spleen, pancreas, thymus, brain, spinal cord, neuronal or retinaleye tissue.
 35. A method of identifying a peptide which permits orfacilitates the transport of an active agent through a human or animaltissue, comprising the steps of: (a) administering in vivo or in situ tothe gastro-instestinal tract of an animal a predetermined amount ofphage from a random phage library or a preselected phage library; (b) ata predetermined time harvesting phage which is transported to the braintissue of the animal to select transported phage; (c) amplifying thetransported phage in a host; (d) repeating in order step (a) using thetransported phage obtained in step (b) and amplified in step (c) andsteps (b) and (c) a predetermined number of times to obtain a selectedphage library containing phage which can be transported from thegastrointestinal tract to the brain tissue of the animal; and (e)determining the identity of at least one peptide coded by phage in theselected phage library to identify a peptide which permits orfacilitates the transport of an active agent through a human or animaltissue.
 36. The peptide of claim 14, wherein the phage is administeredto the gastrointestinal tract and is harvested from brain tissue. 34.The peptide of claim 14, wherein the havesting site is tissue selectedfrom the group consisting of duodenum, jejunum, ilium, ascending colon,transverse colon, desending colon, pelvic colon, vascular endotheliumwhich lines the vascular system, vascular endothelium of the blood brainbarrier, vascular smooth muscle, alveolar, liver, kidney, bone marrow,heart, spleen, pancreas, thymus, brain, spinal cord, neuronal or retinaleye tissue.
 35. A peptide comprising SEQ ID NO: 19 or a fragment thereofwhich contains at least 6 amino acid residues.
 36. A peptide comprisingSEQ ID NO: 21 or a fragment thereof which contains at least 6 amino acidresidues.
 37. A peptide comprising SEQ ID NO: 23 or a fragment thereofwhich contains at least 6 amino acid residues.
 37. A peptide comprisingSEQ ID NO: 25 or a fragment thereof which contains at least 6 amino acidresidues.
 38. A peptide comprising SEQ ID NO: 27 or a fragment thereofwhich contains at least 6 amino acid residues.
 39. A peptide comprisingSEQ ID NO: 29 or a fragment thereof which contains at least 6 amino acidresidues.
 40. A peptide comprising SEQ ID NO: 31 or a fragment thereofwhich contains at least 6 amino acid residues.
 41. A peptide comprisingSEQ ID NO: 33 or a fragment thereof which contains at least 6 amino acidresidues.